Recognition of messenger RNA during translational initiation in Escherichia coli

Revue

BIOCHIMIE. 1984, 66. 1-29

Recognition of messenger RNA during translational initiation in Escherichia colt. E.J. GREN Institute of Organic Synthesis, Latvian SSR Academy of Sciences, Riga, USSR. (Recue le 5-10-1983. acceptre le 25-10-1983)

R6sum6 -- Les aspects structuraux de la reconnaissance par ies ribosomes d ~ . coli des rdgions d'initiation de la traduction sur des RNA messagers homologue font l'objet de !a prdsente revue. Nous discutons dgalement de ia localisation de ia rdgion d'initiation sur le RNA messager, de ses limites, des s6quences nucidotidiques responsables du signal d'initiation et de l'influence de la macrostructure des RNA sur l'initiation de la synthdse protdique. Nous avons rduni la plupart des sdquences nucldotidiques du DNA entourant le ddpart de divers g~nes dE. coli amsi que celles de ses phages~ publides ~ ce jour. Mots-cl~s : initiation de ia traduction / site d'attachement ribosomal / codons initiateurs / S&luence de Shine et Dalgarno / structure du RNA messager.

Summary -- The structural aspects of recognition by E. colt ribosomes o f translational initiation regions on homologous messenger RNAs have been reviewed. Also discussed is the location o f initiation region on mRNA, its confines, typical nucleotide sequences responsible for initiation signal, and the influence of RNA macrostructure on protein synthesis initiation. Most o f the published DNA nucleotide sequences surrounding the start o f various E. colt genes and those o f its phages have been collected. Key-words : translational initiation / ribosome binding site / initiatory codons / S h i n e - D a l g a r n o sequence / mRNA structnre.

1. Introduction Translational initiation in prokaryotes as one of the steps in the multistage process of genetic information realization has been in the highlight of many investigations (for review see [1, 6]). Both the fine mechanism of initiation and the structural aspects of interaction involving individual components of the protein-synthesizing machinery in the cell have been examined. It soon became apparent that translational initiation in prokaryotes and eukaryotes has many divergent features, with respect to both the mechanism and the structural aspects. The prokaryotic system being

less complicated and better understood, especiallv at the initial stage of investigation, has played a leading role in our general understanding of translational initiation; and it still remains the most inves:igated system. However, recent years have seen tremendous progress in the study of translational initiation in eukaryotes, largely due to its key role in the regulation of gene expression. Although in terms of amount and diversity of regulatory, elements, translation is considerably inferior to transcription, the available data nevertheless speak up for the possibility of also controlling gene activity at the translational level, mainly during initiation stage [7, 16].

2

E.J. Gren

According to the current zaotions, translational initiation in prokaryotes, predominantly formed on the basis of the E. coli system, calls for the 30S + IFI + IF3 ~ fMet-tRNA + GTP + IF2 ~ 30S.IFI.IF3 + fMet-tRNA.GTP.IF2 ~-30S.IFI.IF3 + mRNA ~--3OS.IFI.IF3.fMet-tRNA.GTP.IF," + mRNA

formation of a 70S initiation complex involving several steps :

30S.IFI.IF3 fMet-tRNA.GTP.IF2 30S.IFI.IF3.fMet-tRNA.GTP.IF2 30S.IFI.IF3.mRNA

(1) (2) (3a) (3b)

% 30S IFI.IF3.fMet-tRNA.GTP.IF2.mRNA (4) .->t 30S.IFI.IF3.mRNA + fMet-tRNA.GTP.IF2 30S.IFI.IF3.fMet-tRNA.GTP.IF2.mRNA + 50S---* 70S.fMet-tRNA.mRN~.|FI + IF2 + IF3 + GDP + P, (5)

The 30S 6bosome subunit activated by initiation factor IF3 first binds either the initiator fMet-tRNA complex with GTP and another initiation factor IF2 (3a) or mRNA (3b). This fairly unstable intermediate complex produces at the next stage (4) a stable 30S initiation complex. The involvement of the thirizl initiation factor IFI in the formation of 30S initiation complex is still poorly understood. The last step (5) involves irreversible t~mding of the 30S initiation complex to 50S subunit with concurrent release of all initiation factors and GTP cleavage to GDP. The complete 70S initiation complex with fMet-tRNA at the P-site of the ribosome is ready to bind the appropriate aminoacyl-tRNA at the free A-site and nrmeeed t n t h e t r n n ~ l ~ t l n n t~lt~na~t;r~n c t ~ n c The available data indicate that selective recognition of proper initiation sites on mRNA is accomplished at the stage of 30S.IFI.IF3. fMet-tRNA.GTP.IF2.mRNA intermediate complex formation (4). Subsequently, the 30S complex is only fixed by the addition of a 50S ribosome subunit and removal of initiation factors accompanied by GTP hydrolysis. The ribosome covered regions of the template have equal length both in the 30S and 70S initiation complexes, being different in this respect from 40S and 80S initiation complexes in eukaryotes [17]. The present review is not dedicated to the mechanism of initiation and to the unravelling of the role of various components of the proteinsynthesizing system in mRNA recognition or that of fMet-tRNA in initiation. Only those structural features of the template will be discussed that are responsible for the precise recognition of initiation sites on it, Le. determinants of mRNA recognition. The surveyed data will be only confined to E. coli and its bacteriophages, since the present BIOCHIMIE, 1984, 66, no !.

system is, by far, much better studied as compared to other bacteria. However, evidence has been accumulating that translational initiation both in gram-negative and gram-positive bacteria studied so far is mainly the same. Structural differences may exist between bacterial species as to their determinants of translational initiation leading to interspecies translational barriers. But even in such cases the structure of initiation determinants and organizatory principles of initiation signals o , mRNA have many features in common [5, 6, 20-241. Apart from the problems of structure-function organization of the protein-synthesizing machinery and the r~echanisms of genetical information expression in a ~acteriai ceil, the mRNA determinants recogiaized during translational initiation have attracted particular interest over the past years. This is in relation with the problem of expression of heterologous eukaryotic genes within recombinant DNA molecules in a bacterial cell. In order to provide the introduced genes with the regulatory regions for translation necessary for their expression, one has to know the general organizational principles involved and the position of these regions at the start of the structural part of the gene on prokaryotic mRNA.

2. Ribosome Binding Sites When nIRNA, a.q initiation factors, GTP and fMet-tRNA, are incubated with ribosomes under special ionic conditions, the latter are capable of recognizing the initiation region on the mRNA and form a 705 initiation complex. If components responsible for subsequent elongation of polypep-

Recognition of messenger RNA tide synthesis (aminoacyl-tRNA, elongation factors) are deleted from the incubation mixture, the ribosome becomes stuck to the mRNA at the beginning of the gene and as part of the 70S initiation complex protects the template segment it is covering, i.e. the ribosome-binding site, from digestion by pancreatic RNase. The use of this experimental approach allows to isolate the ribosome-bound fragments of mRNA, and, if mRNA is labelled with 32p, to determine their structure by the conventional fingerprinting technique

[3, 4]. Application of this technique has led to the establishment of the primary structure for many ribosome binding sites, first for the genes of RNA-containing phages [25-30] and later for other mRNAs. Moreover, other approaches were also used to establish mRNA structure around the point of initiation including the sequencing of the corresponding D N A (sometimes also mRNA) fragments. Table I presents the structure of DNA and RNA regions around the start of a great many

TABLE 1 Nucleotide sequence around the initiator codon of various E. coli and bacteriophage genes.

A variant of the Shine-Dalgarno sequence complementary to the E. coil 16S rRNA 3"-terminal nucleotides .oAUUCCUCCACU... is underlined (G-U base-pair is marked with a dot) [20, 81]. When known, the starting point of mRNA synthesis lying within the given nucleotide sequence is indicated by an arrow. RNA phases: RI7, A ~25,157,1581

CCG GAA UUC CAU UCC UAG GAG GUU UGA CCU AUG CGA GCU UUU AGU GCC A

C 125,31,34,159,160] (1'2)

UAA AUA GAG(CICC UCA ACC GGG GUU UGA AGC AUG GCU UCU AAC UUU ACU

R 25,161!

AUG CCG GCC AUU CAA ACA UGA GGA UUA CCC AUG UCG AAG ACA ACA AAG

MS2, A "162,163

CCG GAA UUC CAU UCC UAG GAG GUU UGA CCU GUG CGA GCU UUU AGU ACU

C

1125,164

UAG AUA GAG CCC UCA ACC GGA GUU UGA AGC AUG GCU UCU AAC UUU ACU

R 38,125

ACG CCG GCC AUU CAA ACA UGA GGA UUA CCC AUG UCG AAG ACA ACA AAG

L

,12b,1271

UUG UUA AGG CAA UGC AAG GUC UCC UAA AAG AUG GAA ACC CGA UUC CCU

C

27 I(2)

fr,

R

i39,40

ACC CGU GCC AUU CCA ACA UGA GGA AUA CCC AUG UCA AAA UCA ACA /LAG

Q~,

A

2~,165

AGC AGU ACU UCA CUG AGU AUA AGA GGA CAU AUG CCU AAA UUA CCG CGU

C

26,30,36,166

AAA GCG UUG AAA CUU UGG GUC AAU UUG AUC AUG GCA AAA UUA GAG ACU

R

~29,30,167,168

f2,

DNA

G4,

AA AUA GAG~C~CC UCA ACC GGA GUU UGA AGC AUG GCU UCC AAC UUU ACU

GCU UAC UAA CUA AGG AUG AAA UGC AUG UCU AAG ACA GCA UCU

phages:

qbx 174,

A

169,170

TTC ATG CCT CCA AAT CTT .0._GA_G__GC TTI TTT ATG[ GTT CGT TCT TAT TAC

A* !169,170

GAC TGT TGA AGA TTG CTG GAG GCC TCC ACT ATG[ AAA TCG CGT AGA GGC

B

~169-17 i,

CCT TGC TGC TAA AGG TCT AG G AGC TAA AGA ATG[ GAA CAA CTC ACT AAA

C ii69,170-

AAT TAA ATC GAA GTG GAC TGC TGG CGG AAA ATG I AGA AAA TTC GAC CTA

D

i169,170

TGC TGT TCA ACC ACT AAT AGG TAA GAA ATC ATG I AGT CAA GTT ACT GAA _ _

E

~169,170",

GrG CTC GTC GCT GCG TTG__AGG CTT GCG TTT ATG[ GTA CGC TGG ACT TTG

F

i169-17 i

GGG CTT CGG CCC CTT ACT TGA GGA TAA ATT ATG[ TCT AAT ATT CAA ACT

G ~169-173-

GCG CTA GGT TTT CTG CTT AGG.AGT TTA ATC ATG[ TTT CAG ACT TTT ATT

H ~169,170-

TTG TCT CCA GCC ACT TAA GTG AGGTGA TTT ATG[ TTT GGT GCT ATT GCT

J

IL169-171

TCA AAA ATT ACG TGC GGA AGG AGT GAT GTA ATG ITCT AAA GGT AAA AAA

~A7o

CGC AGA AGT TAA CAC TTT CGG __ ATA TTT CTG ATG IAGT CGA AAA ATT ATC

A ~174

AAT ATG CCT CCC ATC AAA CGG AGG CTT TTC ATG TTT AAA GTA CAT TCT

(i) Some bacteriophage preparations have GGA, others have GGG. (2) Uncertainty in (C)CC sequence, CC is presented instead.

BIOCHIMIE. 1984, 66, flo I.

3

4

E.J. Gren A*

GAG TGC TCA AAA ATC TTG GAG GAG TCA ACT ATGIAAG TCT CGA CGT CGC

1174-

CAT GGC CGT AAA AGG CCT AGG GAA TAA AGA ATGIGAA CAA TTC ACT CAA

B 174 C

74-

AAT TAA ATC GAA GTG GAC TGC TGG TGG _AAA ATGIAGG AAA TTC AAT CTC

D

74

CAA CTG ACA CAA ACC ACA AAG GAA ACT GAA ATG[TCT AAA TCA AAC GAA

E

174"

GCG CTC 6CC GTG CTA TCG_AGG CTT CCG T A T ATGIGAA CAC TGG ACT TTG

F

174

CGC GGT CCC ACT erA TTT AAG GAT ACA AAA ATG~TCT AAC GTT CAA ACA

G

174

TCC CTA CTG CAA AGC CAA AAG_G&C TAA CAT ATGI TTC CAG AAA TTC ATT

H

174~

TCC TTC AAC CTC TGA A A ! A A G

j

174~

TTA TCG TCT TCA CTT TTA AGG AGT TAT GTA ATG' AAA AAA TCA ATT CGC

K

1745

CCA CGA AAT TAA CAA GTA CGG A TA TTT CTG ATG AAA CCA AAA ACT ACG

!

fd,fl,Ml3 1 175-17~_

(3)

GA_T TAT CCT ATCI TTT GGC TCT ATE GCT

CGT TTC TTA TTT GGA TTG GGA TAA ATA A~T ATG GCT GTT TAT TTT GTA

II

.175-178j

GGG CTT TTC TGA TTA TEA ACC ~GG GTA CAT ATG ATT GAC ATG CTA GTT

III

~175-178j

]175-178 i (5)

TTG GAG CCT TTT TTT TTG GAG ATT TTC AAC GTG AAA AAA TTA TTA TTC C GTA CTG TTT CAA TTA AAA AAG GTA ATT CAA ATG AAA TTG TTA AAT GTA C AGT TCT TAA AAT CGC ATA AGG TAA TTC AAA ATG ATT AAA GTT GAA ATT

vl

<17>17si

TAA

VII

~175-178 i

ACC GTC TGC GCC TCG TTC CGG CTA AGT AAC ATG GAG CAG GTC GCG GAT

~17~-1781 (~)

zv V

CAT ACT GCG TAA TAA GGA GTC TTA ATC ATG CCA GTT CTT TTG GGT

GTA TTT TAC CCG TTT AAT GGA AAC TTC CTC ATG AAA AAG TCT TTA GTC IX

~175-178j

X [175-1791

~,

Nu 1 1 8 0 '

A 11S03

CGC GCT TGG TAT AAT CGC T G G _ G C G T C A

AAG ATG AGT GTT TT~ GTG TAT

GAA CTG TTT AAA GCA T T T G 6 G G G G G A T

TCA ATG AAT ATT TAT GAC GAT

CTT TCT CTG TTT TTG TCC GTG_ GAA __ TGA ACA ATG GAA GTC AAC AAA AAG CGC TGG ATG AAC TGA TAC CG_G GGT TGC TGA ,GTG AAT ATA TCG &gC AGT

w

ilS0~

TTA CGC CCG TGC CTT ATC CG_G AGA GGA TGA A T G AC~ CGA CAG GAA GAA

B

i180~

GCG ACG CAG GGG ACC TGC AGG ATT TTA TGT IATG AAA ACG CCC ACC ATT

C

~,180i

GGC TGC GAC AAT CAA CAG AGG __AGG . AGA AGAtGT( ACA GCA GAG CTG CGT

D

180

TTG CTG AAC ACA CCA GTG TAA GGG ATG TTT IATG ACG AGC AAA GAA ACC

E

180

CCT GTG CGG CTT TTT TTA CGG TTT TTT {iATG TCG ATG TAC ACA ACC ., CAT .

F

I

180:

F

II

180

i J

CGG GGC CAT TGT TTC TCT GTG GAG GAG TCC iATG ACG AAA GAT GGA CTG i CCT GGC CAG AAT GCA ATA ACG GGA GGC GCTIGTG GCT GAT TTC GAT AAC k

z ~18oi

CGr TAA CCG TCG CCG CTG AAA GGG G GA TGTiATG GCC ATA AAA GGT CTT

u

1so!

V

180~

GCA GCA TCA ACT GAG GAT GGT AAT AAA GCG"ATG AAA CAT ACT GAA CTC .... i ATG TCA TTA CCT ATG AAA TGT GAG GAC GCTI ATG CCT GTA CCA AAT CCT

G

180-~

T

180 ~

GTC ACC GCC AGT TAA TCC GGA GAG TCA GCGI ATG TTC CTG AAA ACC GAA i CGG GCC CGC AGA GCC TGT TTC TGC GGG AAA 1GTG TTC GAC GGT GAG CTG

H

~180"

TAT CAG AAG GGA TCG CAG ,GAG ,GAG TCC GGT ATG GCT GAA CCG GTA GGC

M

180

TGA TGG TGG CCT GTT CTC CGG AGG TGG ACG ATG AAG ACC TTC CGC TGG

L

!80

CAG CGC AGA GTT TGA ACA GGT GGT GAA CTG ATG CAG CAT ATC CGG CAG

K

180

TAT CTC CGG TGA GCC GGA GGC TAT TTC CGT 4TG ITCG CCG GAA GAC TGG

I

180

ACT GAG CAA ACG AGA GAG GTA CAC CGA CAA IATG GCA GCG ACG CAC ACA

J

180

GGC GGT TTT GTC ATT TAT GGA GCG TGA GGAI ATG GGT AAA GGA AGC AGT

cro

180,311-314

GAT AAT GGT TGC ~ATG TAC TAA GGA GGT TGT ATG GAA CAA CGC ATA

(~) The bracketed nucleotide in the A(A)T sequence is missing in phage fl and MI3 DNA. (') Sequence CCA instead of CAA has been found in phage fl and MI3 DNA. (~) Sequence ACA instead of AAA has been found in phage fl and Mi3 DNA.

BIOCHIMIE, 1984. 66, n ° 1.

ACC

Recognition of messenger RNA c II

150,311,312

ATC AAT TGT TAT C T A _ A G G A A A

5

TAC TTA CAT ATG GTT CGT GCA AAC AAA

0

lZ0,311,315

TCA TTA CTG GAT CTA TCA ACA GGA GTC ATT ATG ACA AAT ACA GCA A~A

P

1~0

AAA CAC AGA CTG GAT TTA CGG GGT GGA T C T A~ T G -

ten

le0

AAA AAC ATC GCC GCA

AAG TGT!ATG ~ACG GGC AAA GAG GCA

AGC TAA GTT CGG ACT GA A A G G A G C

,:~n A

179

ACG GGT ATT GGC GGT ATA TGG AGT TAA A A G A T G

ACC ATC TAC ATT ACT

n:n B

I?~+~ ],~0

AGG CCT TTT TAT TTG GGG GAG ACG G~I G T C A T C

AAA AAA CTA ACC TTT

n:n C

179,1~0'.

GTG GAA AGC GAG ATG G G G A G A

nln D

179,1m0

GCT TGA TTT CGA CTT COG GAG GGA AGC TGC ATG ATG CGA TGT TAT CGG

nln E

179,1~0

GTT ACC ACT ACC GCA GGA AAA GGA GGA CGT GTG GCG AGA CAG CGA CGA

nln F

179,1~0

GTG GCT AAG ACG TCG TGC GAG GAA AAC AAG GTG ATT GAC CAA AAT CGA

nln G

179,150

ATC CGA ATA GCT CGA TGC ACG AGG AAG AAG ATG ATG GCT AAA CCA GCG

n~n H

179,160~

AGA CCT GCG AAA TAG CAG AAG TGA GGC C G C ATG ACG TTC TCA GTA AAA

q

AAC ATT GAT TCA GGT A C A G G G

.iB0,I~2,3151

TAA GAC ATG AAG ATG CCA GAA AAA

GCT TCG C'FG CTA AAA AAG CCG GAG TAG AAG ATG GTA GAA ATC AAT AAT

i~0

Rz

AGA AGG CGC ATG AGA CTC GAA AGC GTA

TAG AGC AAA TCC CCT TAT T G G G G G

S .i~0,3181 R

CAG GGC TGC:ATG ATA AAT GTC G fT AGT

AGC GGG CGG ~AC ~G~ CAG AGA GAT TGA TGT ATG AGC AGA GTC ACC GCG

~0+

c l 53,54,1B0i

(7)

ATT TAT CCC TTG CGG TGA TAG ATT TAA CGT ATG AGC ACA AAA AAG ~AA

rex A i160,1~5:

CAG ~AC ATA ACA TAG TAA ATG GAT TGA ATT ATG NAG ANT GGT TTT TAT

rex B ~l~0,1B5.

AAA GTT AAG GAT TAA TTA TCA GGA GTA ATT, ATG CGG A_A,C AGA ATe ATG

+ (8)

N180,183

GCC GTA AGT GCG ATT CCG GAT TAG

ra[

i~0,151

ATG TGA TGG AAC .EAT ACC AGG &CT ATC CGT[ATG i ACT ACG ACT ATT GAT

ssb

150,151

GCG ATT TTT TAT CTG TGA GGA TAT GAA CAGIATG[ TCA AAC ATC ~AA AAA

i

cllt

l~O

kil

(~)

I~0

(~)

I

TTT TCG TTA TGT ATA A A T A A G + G A G ACA TAC TTA ATC AGC CAG GAG TCC CAA AGAIATG|GAT CAA ACA CTT ATG TAr TTT CAC AGC TAT TTC AGG_AGr TCA

le0,1B1 ~×o

l~O

Ea 22

130

Ea ~.5

i~0

xis

b6,160

int

66,180

~ . . . . . . GCT TAT TAC ATT I , CGAIATGIAGT ACT GCA CTC GCA GGT TGA TAT TGA TTC AGA GGT ATA AAA I I CCG GAC ATT ATC ACA GGA AGC CGC AGA GCA GAA GGT GGC AGCIATGIACA | GGA ACT TAT CAG T~A ACA GAG A G G ! T C

GAA 3TGIAGC GAA ATT ~AC TCT

GAP, ACG ACA TAC ATT GCA AGG AGT TTA TAA ~TGI AGT ATC AAT GAG TTA ...... i TTT CTT GCG TGT AAT TGC GGAGGAC TTT GCG ATG~ TAC TTG ACA CTT CAG |

Ea 59

i~0

TAA CAG GTG GCC TTT TGA AGA GGA TCA GAA ATGIGGA AGA AGG CGA AGT ! GGT TAT TTT AAT AAA A T T A A G GGT TAC TAT ATG] TIG GAG TTT AGT GTT

Ea 31

150

GAT AAT CAA tAG AGA TGA AGG TAA AGT ACA ATG| AAA AAA CTA CCT CTT

Ea 47

1~0

AGC ATG CAA CCT ATC ~AA ATG+G& Q AGT TTT ATG' ACT AAA AAA CCA TGG

!

434,c I cro Mu,

A

317

t

AAG ATT GGG GGT AAA TAA CAG A_GG T_GG CTT ATG AGT ATT TCT TCC AGG

317

AAT ACA AGA AAG TTT GTT GAT GGA GGC GAT ATG CAA ACT CTT TCT GAA

1.199

GGC CTA GCC GAT ATC AAG CAG GTG AAT AAC ATG GAA CTT TGG GTA TCA

199

AAG TCA ArT TGG TGT TCG CCG CAA GAA ATTIATGIGCT GCT GAT GGT ATG

.

c I

.

.

.

Il

.

!

e II

199

t

ATA GAT TAC AAA ACT TAG GAG GGT TTT TAA I ATGI TGT TCC AAC GAA AAG

TT, 0.3 J

0.4

_131,186,1871~

0.5 A

186 !

i

AGT CGA GGA GTA CGA_. .GGA . . . GGA TGA AGA GTAIATGITCT ACT ACC AAC GTG TAT GAT TAT CAC TTT AGT TAT G_AG G_GA GTAIATGITAT ATG CTT ACT ATC

_

(7) Promoter P,t,a directed transcript begins directly from initiation codon, whereas a long nontranslating part of template is synthesized from promoter PRt; (") Another variant of the gene starting point is presented [1811.

BIOCHIMIE, 1984, 66, n ° I.

E.J. Gren

0.6

18b

0.65

186

O. 7

TAT CAC TTT ACT TAT GAG GGA GTA ATG TAT ATG CTT ACT ATC GGT CTA

1~6

O. 5 B

CAT AGG AAT CAT C~A AGG GGC ACT ACG CAA ATG ATG AAG CAC TAC GTT ACC

GCA AGA TTA ACA AGA TAG GTT CCG GCT ATG ACA GAA CGC ACT GAT

TCT CAT AAC GAA CAT AAA GGA CAC AAT GCA ATG AAC ATT ACC GAC ATC

J86,

1186,189-191:

GTA CGA rTT ACI AAC TGG AAG AGC CAC TAA ATG AAC ACG ATT AAC ATC

l.I

186,188"

TTA AAG AAT TAC TAA GAG AGG ACT TTA AGT ATG CGT ~AC TTC GAA AAG

1.2

186,1S$

CTC ACA AGC GTA GCT G G G A G G

!

1.3

GTC AGT AAG ATG GGA CGT TTA TAT ACT

TAG TCA TTT AAC CAA TAG GAG ATA AAC A T T ATG[ATG AAC ATT AAG ACT

186

i

1.4

186

GGC CTT TCT GCG TTT ATA AGG~_ AGA CAC TTT IATG TTT AAG AAG GTT GGT _

i. 5

is6 ~

TAA

1.6

186

CTT AAT ACG ACT CAC TAA AGG AGA CAC TAT ATG[TTT CGA CTT CAT TAC _

1.7

186

GTT

ACT CAA ACG AAT CAA GGA GGT GTr CTG ATGIGGA CTG TTA CAT GGT

i. s

is6;

AAC

TCT TTG AGA AAC ATA AGG ATA AAT GTT ATGICAT AAC TTC AAG TCA

TAC GAC TCA CTA _AAG GAG GTA CAC ACC ATG ATG TAC TTA ATG CCA

[

I

2

186

2.5

186

ACC AAA CGA AAC CTA AAG GAG ATT AAC ATT ATGIGCT AAG AAG ATT TTC

le6

CGA GGA AGC AGC GAA GAC GGA GAC TTC TAA

~iGIGAA CTG CGG GAG AAA

CTA TTT GTG ATA TAC GCA AAG GGA GGC GAC

~TGIGCA GGT TAC GGC GCT kTG[GCT CGT GTA CAG TTT

2.5 3

TGA ACT TTG GAA ATC GAG AGG TCA ATG ACT ATGITCA AAC GTA AAT ACA

lSb

3.5

166

TAG ATT AAA AAG GAA AGG AGG ~ A

GAA ATA

3._~

1~6

TTT CCC TTT GTT CGC ATT GGA GGT CAA ATA ETG{CGC AAG TCT rAT AAA

4 A .186

CGT GGA TTA ATA GAA CTA GGA GGG AAT TGC ~TG[GAC AAT TCG CAC GAT

4 B

156

TCA AAA CGG AAA CCC TCA GGA GGT AAA CCA ~TGIACT TAC AAC GTG TGG

133 "

CTG TAG TTC AAC TTT AAG GAG ACA ATA ATA ATGIGCT GAA TCT AAT GCA

9 10

133

17

133

T4, r ii B

TTT GTT TAA CTT TAA GAA GGA GAT ATA C A T A T G I G C T

(b)

42,64,193

ACT CTC TTA GAT TTA CTT TAA GGA GG'r CAA

AGC ATG ACT GGT

ATGIGCT

AAC GTA ATT AAA

GAA GGC CCT TGC GGC CTA ATA AGG AAA ATT ATGITAC AAT ATT ~AA TGC

~2

!0a

36

195

TAA TTA aaT TAa aTT AAA Aa~ eaa aTa Aa~ ATGITTT ~ A CGT AAh TCT I ATA AAT ACT AI'T AAA ATA AAG GGG CAT ACA ATG[GCT GAT TTA A~A GTA

37

195

TCA AGA TTT CGG CTA TTA TTA AGA GGA CTT ATGIGCT ACT TTA #AA CAA

38

195

ATA AGG AGA GGG GCT TCG GCC CTT CTA AAT ATG[AAA ATA TAT CAT TAT

45

196

TTA AAT CTC ArT TGA ATT G AA GGA AAT TACIATGIAAA CTG TCT AAA GAT

57 A

197

67

19~

I

Escherichia

coli:

ala S

200

ATA TTT CGT TAG CTT GAT TTC AGG ATA ATTLATG'ACC AAG AGC ACC GCT GCG GGC CGT TTT GTA TGG AAA CCA GAC CCT ATG TTC AAA ACG ACG CTC

amp C

201-203

ara B

204

ara C

205,206

TCT TCT CTG AAT GGC GCG AGT ATG AAA AGT AFG GCT GAP, GCG CAA AAT

arg C

207

CAA CCA GAC ATA AGA AGG . . . . TGA ATA GCC CCG ATG TTG AAT ACG CTG ATT

arg E

207

GAT ACT ATC ATG ACC AGA GGT GTG TCA ACA ATG AAA AAC AAA TTA CCG

A CCC GTT TTT TTT GGA T G G A G T

GAA ACG ATG GCG ATT GCA ATT GGC

(~) The appropriate DNA sequence in phage T3 and T7 is identical except for the nucleotides prior to the initiator codon; CCC in T3 instead of CAA in T7 [192]. (~) The most frequent nucleotide sequence around the initiation codon obtained by computer processing of structural data for initiation regions (nucleotides CTCAA should be regarded as continuation of the main line).

BIOCHIMIE. 1984, 66, n ° l.

Recognition of messenger RNA arg F

20~

aro G

209

AAT AAG TCC CGr TCG CCA TGC GAG CAT AAA ATC TCC CAT TTA TAC AAA TAA GAT AAG TAT GGC AAC ACT GGA ACA GAC ATG AA]

CAT AAG CAA CAG GAC GCA GGA GTA TAA ~ A

211

bla

ATG AAA ACC GCT TAC AT1

ATG CTT CAA TAA TAT TGA AAA AGC AAG AGT ATG AGT ATT CAA C~T TTC

212,213

bio A

IA] CAG AAC GAC

CGT AGA AGC AAC AAA TTT CTG AGA CTY GTA ATG ~4C AGA ACT GAC G ~ .

aro H 2 1 0 asn A

7

AAT CTT TTC AAT TTG GTT TAC AAG TCG ATT ATG ACA ACG GAC CAT C

21~

bio B 1214

TAA CAA CAA AAA ACA CG~ TTT GGA AGC CCC ATG GCT CAC C

cat

TCG AGA TTT TCA GGA GCT AAG GAA GC~ AAA ATG GAG AAA AAA ATC ACT

215,21b

clo DF 13 "218

ATT GTT TTA AAT GTC AAA GAG GAA AAC GAT ATG AGT GGT GGA GAC GGT

clo DF 13H

TGC CGA AGC CTC TGA ATA CAA GGA AGG TAT ATG A ~

321

AAG GCA AAG GCT

clo DF 13 iman 217,321

AAC ATT AAA AAA TAT CTT TAA GAG GTA ~4T ATG GGG CTT ~

col El

ATT GTT TTA AAA GTC AAA GAG GAT TTT ATA ATG GAA ACC GCG GTA GCG

crp

21S-220

TTA CAT

GAG AAA GCT TAT AAC A~A GGA TAA CCG CGC'ATG GIG CTT GGC .&~A CCG

221,222

dna A 2 2 3

TTA TCG ACT TTT GTT CGA GTG GAG TCC GCC GTG TCA CT~ TCG CTT TGG

dna G

TCG TTT ATG AAT TGC TAA AAA TCG_GGC CCT ATG GCT GGA CGA AYC CCA

-224.

dna N 223.

AAT CTG CTG TTA CAG GTT GCT GAC GGT ACG GTG TCG CTG ACC GGT ACT

eli A ;225~

TCT TCT TGT ATG ATA TAT AAG TTT TCC TCG ATG AAA AAT ATA ACT TTC G TGA CAT ATA TAA CAG AAT TCG GAA TGA ATT ATG AAT AAA GTA A . ~ TGT

eli B 228,322! endo EcoRI

(i0)

2271

TTT ATA AAA TAA C%G TGG 4AA CAT GG6 TTC ATC TCT ~ T

AAA AAA CAG

env Z [-22b~

TTT AAT AAA GTC CTC GCG TAC GAA GTG CGT ATG TTG ATG ACC GAC AAA

~st i ~229~

TAT CCG TGA AAC AAC ATG ACG GGA GGT ~ C

fnr

ACA AAT ATC AAT TAC GGC TTG AGC AGA CCT ATG ATC CCG GAA AAG CGA

~230= ..

fol

GGC GAC AAT TTT TTT ]AT CGG GAA ATC TCA ATG ATC AGT CTG ATT GCG

231,232

fol (R366)

ATG AAA AAG CTA ATG TTG

233,234

GCC ATA ATA GAT TCA CAA GAA GGA TTC GAC A~G GGT CAA AGT AGC GAT

frd A

235

TGT GGG ATA AAA ACA ATC TGG AGG AAT GTC GTG CAA ACC TTT CAA GCC

frd B

23b

CGG AAG CAG CCA ATA AGA AGG AGA AGG CGA ATG GCT GAG ATG AAA .~AC

142

TGA ACG CCT AAA AGA ~AA ACG AGG g~A CAA ATG GCT CGT ACA ACA CCC

gal E " 2 3 7 , 2 3 S

TTT CAT ACC ATA AGC CTA ATG GAG CGA ATT ATG AGA GTT CTG GTT ACC

gal K

155+

ATC CAT TTT CGC Gk&. TCC GGA _CTG TAA GAA ATG AGT CTG AAA

gal R

23g+

fus

ATG TgR GCG TTT ACC CAC T ~

GG] ATT TTC ATG GCG ACC ATA AAG G~]

CCA CAG GGA TAT CCC GAT TAAGGA %CG ACC ATG ACG CAA TTT gAT CCC

gal T

240~

his

G

241

GTT CAG ACA GGT TTA AAG AGG AAT AAC AAAATG ACA GAC . ~ C ACT CCG

his L

241

TCA GTT GAA TAA ACA TTC ACA GAG ACT TTT ATG ACA CGC GTT CAA TTT

his S _242

TTC ATT TTT ATA TTC AGA AAG AGA ATA AAC GTG GCA AAA AAC AT~ CAA

ilv B ~243

AAA GTC GGC CCA GAA GAA AAG GAC TGG AGC ATG GCA AGT TCG GGC ACA

ilv L (B) gly

+243,244

TTT TAT TGT TAG CTG ACT CA__G GAG ATG CGG ATG TTA AAG CGT GAA ATG

A i~4

ilv E G

ilv

L (GEDA)

inf C kan lac a

TCC GCG CCT GAG CGC AAA__AGG AAT ATA AAA ATG ACC ACG .,-LAG AAA GCT

245

ilv

ACC GTT TTC GGT TAT CCGGGT GGC GCA ATT ATG CCG GTT TAC GAT GCA

.2 4 5

58. 24S ~57~

lac i r~0 ~

TTG TTA AAC ACA AAA CCA ACA AGG TCC CCA ATG ACT ACT TCC ATG CTC

245-247

TCC TTC G~R CA,~ GAT CCA AGA AAA+GAC AAA ATG ACA GCC CTT CTA CGA CGC AGT CTT AAA CAA TTGGAG GAA TAA GGT ATT AAA GGC GGA &&A CGA GCT TAC ATA AAC AGT AAT ACAAGGGGT GTT ATC AGC CAT ATT CAA CGG CGA CCA ACA TAT CAT AAC GGA GTG ATC GCA TTG AAC ATG CCA ATG ACC

G GAA GAG AGT CAA TTC AGG GTG GTG AAT GTG AAA CCA GTA ACG TTA

('") Sequence GGA is also presented instead of GAA [322].

BIOCHIMIE, 1984, 66, n ° I.

8

E.J. Gren lacy

CTG CCC GTA TTT CGC GTA AGG AAA TCC ATT ATG TAC TAT TTA AAA AAC

~5,57"

lag z

GCG GAT AAC AAT TTC ACA CAG GAA ACA CCT ATG ACC ATG ATT ACG CAT

249,2501

lam B 18S,251,252-

AAA AAG AAA AGC AAT GAC TCA GGA GAT AGA AFG ATG AT]' ACT CTG CGC _.

leu A

GGC GCT CTA AA~ GAG ACA _. AGG ACC CAA ACC ATG AGC CAG CAA GTC ATT

2_5 3

leu L

253

AGC ATA TCG CAT TCA TCT GGA GCT GAT TTA ATG ACT CAC ATC GTT CGC

lex A

'254,255,262

GCA TAA CTG TAT ATA CAC CCA GGG GGC GGA ATG AAA GCG TTA ACG GCC

Ipt

GAG ATT AAC TCA ATC TAG AGG GTA TTA ATAfATG '~ A

256,257

GCT ACT AA~ CTG

mal F i25S

CGA GCA CTT CAC CAA CA_A_GGA CCA TAG ATT ATG AAA ATA AAA ACA GGT

mal K

,25b,2591

GAT GAC AGG TTG TTA CAA AGG GAG AAG GGC ATG GCG AGC GTA CAG CTG

ma[ P

[260~

TGT TGA AAA TCT AAG AAA AGT GGA ACT CCT ATG TCA CAA CCT ATT TTT

mal T

i260j

meth EcoRI sol A

TCC TCA TTT TCC ACA GTG AAGTGA TTA ACT ATG CTG ATT CCG TCA AAA ATA TTT TTT ATT TTA ATA AGG TTT TAA TTA ATG GCT AGA AAT GCA ACA

\227j

CGC TGA GCT CCA GAT TTT GAG GTG AAA ACA ATG AAA ATG AAT AAA AGT

i252J

ndh

i'56i

CAA TTG GTT AAT AAA TTT AAG GGG GTC ACG TTG ACT ACG CCA TTG AAA

neo

!261i

GAT C ~

GAG ACA GGA TGA GGA TCG TTT CGC ATG ATT GAA CAA GAT GGA

omp A 2 6 3 !

CGT ATT TTG GAT GAT AAC GAG G_CG CAA A,-XA ATG AAA AAG ACA GCT ATC

omp C ~2641

AAT AAA GGC ATA TAA CAG AGG GTT AAT AAC ATG AAA GTT AAA GTA CTG

omp F

~65,2661

TCA TAA AAA AAA CCA TG~_GGG TAA TAA A T A A T G

omp R

~28~

AAT TGT TGC GAA CCT TTG GGA GTA CAA ACA ATG CAA GAG AAC TAC AAG

phe A

267

CC7 TTT TAT TGA TAA CAA AAA GGC AAC ACT ATG ACA TCG GAA AAC CCG AAG TCA CTT AAG GAA ACA AAC ATG AAA CAC ATA CCG TTT

phe L ~267] pho A

ATG AAG CGC AAT ATT

TTT AAT GTA TTT GTA CAT GGA GAA AAT AAA GTG! AAA CAA AGC ACT ATT

~266,269 i

pho E ~316J

TTC GCG CGT TAA TTA AAA TCA GGA ATG AAA A T G AAA AAG AGC ACT CTG

pit

TTA AGA TAF ACA GAA TGA TGA GGT TTT T T T ~ A T G AGA CTC AAG GTC ATG

i270,271 i

pot A ~72~

CCC ATA ;.I 2 TGA TAA ACA GGC ACG GAC ATT IATG: GTT CAG ATC CCC CAA

pro A i316~

ATT TTT CCT AAA ATT GAA TGG CAG AGA ATCI ATGI AGT GAC AGC CAG ACG

pyr B

273,319

(Ii)

G C GCT TTT TTT TAC CTA GGC AGG AGA T~A AAG~ATG'GCT AAT CCG CTA TAT

pyr I ~274 ~

CGC GAT C~G GTA CTG T AA_G_G_G__GAA ATA GAG ATG ACA CAC GAT AAT AAA

pyr L ~273,319"

ATA ATG CCG GAC AAT TTG CCG GGA GGA TGT ATG GTT CAG TGT GTT CGA

ree A

CCG GTA TTA CCC GGC ATG ACA GGA GTA AAA ATG GCT ATC GAC GAA AAC

,275 ~

rho ~320~

TTT ACC CCA AGT TTA AGA ACT CAC ACC ACT ATG AAT CTT ACC GAA TTA

rpl A

TTC ATG GGC CTG GTA GTG GAG GAC TAA GAA ATG GCT AAA CTG ACC AAG

rpl C

276" 277

AGG TCA TTG AGC GAT TGA GAG GTT GAA ACA ATG ATT GGT TTA GTC GGT

rpl E 1 2 :

CGA AAC TAT CAA GTA ATT T G G A G T

rpl

TTC TCT GGC A~A CAT CCA GGA GCA AAG CTA ATG GCT TTA AAT CTT CAA ..........

J 1276'

rpl K

AGT ACG ATG GCG AAA CTG CAT GAT

276

GGC GTF ATT ACC CAA CTT GAG GAA TTT ATA ATG GCT AAG AAA GTA CAA

rpl L 2 7 6

AAC TTA TTC TGA TAT T C A _ G G A ACA ATT TAA ATG TCT ATC ACT AAA GAT

rpl N :278

G TAG TTG ACA TTA G C G G A G

CCT AAA ATG ATC CAA GAA CAG ACT

rpm B i279~

TCG AGC TAA TTT GAT TTT TGG AGA ATA GAC ATG TCC CGA GTC TGC CAA

rpm G !279

AAA GTA CTA AGT ACT TAG AGG AAA TAA ATC ATG:GCT AAA GGT ATT CGT

rps A ~280

TGG AGT TAA ATA TAA ACC TGA ___ AGA TTA AAC ATG!ACT GAA TCT TTT GCT

rps B 1323

AAC CCC AAC TTT TAT ATA GAG GTT TTA ATC ATGI GCA ACT GTT TCC ATG

rps G !142

ACA ATC CTG AAT TAA CAA CG_G A G T ATT TCC ATG ICCA CGT CGT CGC GTC

('~) A somewhat modified structure [319] with TGC instead of "IAC, CCA instead of CTA, and an insert indicated by A

BIOCHIMIE. 1984, 66, n ° I.

Recognition q[" messenger RNA

9

rps

J

277

CGT TTA TAA AAT AAT TGG AGC TCT GGT CTC ATG CAG AAC CAA AGA ATC

rps

L

L42~ 27b

GAA GCA AAA GCT AAA ACC AGG AGC TAT TTA ATG GCA ACA GTT .-'_%C CAG

rps

M

2~ 1

AAC GTA CAT ATT AAA TAG TAG GAG TGC ATA GTG GCC CGT A]A GCA GGC

rps

T

55

TCC ATA TAG AAC ACA TTT G.GG AGT TGG ACC TTG' GCT AAT ATC AAA TCA

rpo

A !~282 :

rpo

B 127b,283;

r p o

C

rpo D

AGT AAA GCT TAG TAC CAA AGA GAG GAC ACA AT(] CAG GGT TC'f GTG ACA (12)

CGT CGA CTT GTG AGC GAG CTG AGG. AAC CCT ATG GTT TAC TCC TAT ACC

!284,285,

GA'i

286 .

TCA TGA AAT AAG TGT GTG GAT TAC CGT CTT ATG GAG CAA A-4.C CCG CAG

FGT GCT AAC TCC GAC GGG AGC AAA TCC GTG AAA GAT TTA I I A

AAG

rop

1!287"

CAG AAA TTC CCC CTT ACA CGG AGG CAT CAA GTG ACC AAA CAG GAA AAA

ssb

', 285

TCA GAA CGA TTT TTT TCA GGA GAC ACG AAC ATG GCC AGC AGA GGC GIA

tel

(Tn 10)

tet

(pBR322)

thr

A

291

T I T TCG ACC AAA GGT AAC GAG GTA ACA ACC ATG CGA GTG TIG A-AG TTC

thr

B

292

GTA CCC TCT CAT GGA AGT TAG GAG TCT GAC ATG GTT .-tAA GTI IAI- GCC

thr

i2B9 290

CAC TCC CTA TCA GTG ATA GAG AAA AGT GAA ATG AAT AGT TCG ACA AAG TTA AAT TGC TAA CGC AGT CAG GCA CCG TGT ATG AKA TCT AAC A_AT GCG

C

292

ACG GCG GGC GCA CGA GTA CTG GAA AAC TAA ATG AAA CTC TAC AAC TqG

thr L

291

CAG ATA AAA ATT ACA GAG TAC ACA ACA ICC ATG AAA CGC ATI AGC ACC

tna A

i293

CTG TAA TTA AAA TAA ATG AAG GAT TAT GTA ATG GAA AAC TTT AAA CAT

tnp A

213,294

TTA AAA AAG CCG TCA GGC AGG GAG GCC GAT ATG CCC GTT GAT TTT TTG

tnp R

1213,294

TGT CTG ATA TTC GAT TTA AGG TAC ATT T T f ATG CGA ATT T T I GGT I A I

tra T

295

TGA CGT TTT ATT CAA TAT GAA GGA ACA TTG ATG AAA ATG AAA AAA TTG

trp A

153,296

CGA TAT TTT G~A AC,C aCG AGG GGA AAT CTG ATC, G.~_A CC,C TAC C~A I C I

trp B

',296,297

G AC GCT GCG CGC ATA TTA AGG AAA GGA ACA ATG ACA ACA TIA CTT AAC

trp C

151,297

GTC ACC GCA CTG GCG GCA CGA GGG TA-A ATG ATG CAA ACC GTT TTA GCG

trp D

2t~b, 2~)7

CAC CGC GCA TCA TGC ACA GGA GAC T I T CTG ATG GCI GAC ATT CTG CTG

tr;: E

Lq',JOU, ~()]

CTT TTT TTT GAA CAA AAT TAG AGA ATA ACA ATG CA-% ACA CAA AAA CCG

trp

2 q ' - 31~()

ACG CA-A GTT CAC GTA AAA AGG GTA TCG ACA ATG A-AA GCA ATT TIC GTA

L

I_-p

TGC TTC CCC CGC T:'_~ CAA TGG CGA CAT ATT ATG GCC CAA CAA TCA CCC

trp ~

T I C CGC AIA GCG AAA ATC AGG AAT CGA AAA ATG ACT A-AG CCC AIC G I I

Y+.-

CCG GGC AGA TCA CAT CTC CGA GGA TTT TAG ATG GCT GAA ATT ACC GCA

323

t~f tuf A

31J5

GGG GAG

tuf

B

]Oh

CGA TTT ATC CGT GTC TTA GAG GGA CAA TCG ATG TCT AAA GAA AAG TIT

unc

A

31)7, 3()"

GAC GIC TTG CAG TCT TAA GGG" GAC TGG AGC ATG CAA CTG A-AT ICC ACC

urn_ B

307, ~()~

TGT AAT

urn_ C

3(.lq

AAA ACT TTA ACG CCT TA-A TCG GAG GGT GAT ATG GCA ATG ACI TAC CAC

unc

309

T]A AAC AGG TTA TTT CGT AGA GGA TTT AAG ATG GCT ACT GGA AAG ATT

307,3L)~

CGT TTT AAC TGA AAC AAA CTG GAG ACT GTC ATG GAA AAC CTG AAT AIG

D

unc

E

unc

F

AGC ACA ATA GTA AGG AAT ATA GCC GTG TCT A-AA GAA A-AA TIT .

IAA C.~A CAA A_GG_G/A A:%A GGC ATC ATG GCT ]CA GAA .
_

_

]O7, ~ ) ,

AGA ACG TTA ACT AAA TAG AGG CAT TGI GCT GrG AAT CTT AAC GCA ACA

uric G

3(}0

GGC

unc

H

3t)7, 3(2_"

TGT GGC

unc

i

]u7

GAC

31 )

TGC CCG CCC AAC TCC TTC AGG TAG CGA CTC ATG AGT AAA CCG TIC AAA

uvr

B

Co n s t. n.,, u .'-,

5!)

AGG CCG CAA GGC ATT GAG GAG AAG CTC ATG GCC GGC GCA AAA GAG TGA ACT GTA AGG AGG GAG GGG CTG ATG TCI GAA TTT ATT ACG

ACG CGG CAT ACC TCG A=AG G G A _ G C A

GGA GTG AAA AAC GTG ATG TCT

T T - T TAA AAA TTA AGG AGG TAT ATT ATG AAA AAA ATT AAA AAAAA T A ATA A CT C -CTC A-A G

(+:) Sequence from [276] contains insert CGT between nucleotides 27 an `+ 28 prior to the initiation codon AT(;.

BIOCHIMIE.

1984, 66, n ° I.

i0

E.J. Gren

genes of different origin (single-stranded RNA phages, single- and double-stranded DNA phages, E. coli). The nucleotide sequence given table ! is longer than the corresponding ribosome binding sites derived from 70S initiation complexes during pancreatic RNase digestion, amounting commonly to 30-36 nucleotides. Initiator codon is positioned approximately in the middle of the ribosome binding site, closer to its T-end and is surrounded by a variable nucleotide sequence. The latter has long resisted all efforts to identify any similarity features indicating possible involvement in the formation of a translational initiation signal. Are all the determinants necessary for normal initiation located within the ribosome binding site ? This issue has been approached by reassociation of the ribosomes with the ribosome binding sites extracted from initiation complex and other segments from this mRNA region. Attempts to reassociate ribosome binding sites for three phage RI7 genes with the ribosome yielded conflicting results. Despite their comparable size, only the ribosome binding site of the A-protein gene (I) appeared active, while the other two -that for the phage coat protein (II) and replicase (VIII) - - were practically inactive under the same experimental conditions [31]; (table II). Contrary to fragment IX from the initial part of phage MS2 replicase gene, active in initiation [32, 33], the fairly long segment (59 nucleotides) from phage RI7 RNA (III), comprising the ribosome binding alt~ ut tnc t;uat protein gene, was inactive [34]. The ribosome binding site of phage Q[i coat protein gene (vI) and its derivative elongated to the 5"-end (V) also failed to act as templates in initiation [35]. However, further elongation of the latter fragment towards the 5"-end as far as 97 nucleotides prior to the initiator codon (IV) led to the appearance of ribosome binding activity [35, 36]. Such behaviour of free mRNA fragments bearing ribosome binding sites may be due to two reasons • l) It is possible that the spatial structure of short inactive fragments is somewhat different from the structure of long active ones; in this case, the structure is closer to that of the active conformation of the initiation region within the native mRNA. As we shall see, the spatial structure (secondary and tertiary) of mRNA plays an important part in the activity manifestation of initiation regions. 2) On the other hand, it can be assumed that during initiation the ribosome interacts with a longer part of the template beyond the ribosome binding site covered by the riboBIOCHIMIE, 1984, 66, no I.

some. In this case, the ribosome binding site is only part of the true initiation region containing all the determinants responsible for ribosome binding. It has been demonstrated that it is not the entire template segment covered by the ribosome that is essentially involved in initiation complex formation. It appears that phage RNA fragments terminating directly at the initiator codon, i.e. devoid of all the ribosome covered part downstream from the AUG codon, are capable of ribosome binding into initiation complex, their activity being equal to that of the fragments containing this part of template. This has been exemplified by initiation regions both of phage Q[~ coat protein gene [35, 36], (cf. Vl and VII, table II) and of MS2 replicase gene [33, 37, 38], (cf. IX, X, and XI, table II). Consequently, the part of ribosome binding site to the right of the initiator codon, although covered by the ribosome within initiation complex, plays no essential role in initiation signal formation on the template. The use of phase MS2 replicase gene enabled to further pinpoint the boundaries of initiation region. In this case, the presence of only 33-35 nucleotides prior to the AUG codon did suffice for the fragment (XII) to exert its activity, although somewhat lower than in (X). However, a further shortening of the 5'-terminal part of the fragment to the left-hand boundary of the ribosome binding site yielding fragments (XIII) and (XIV) led to an abrupt drop in template activity [37,38]. Possible spatial structure variations among fragments of different length, which might account for such dramatic differences in activity, are apparently ruled out in this case. Therefore, it appears more plausible that the observed phenomenon is due to differences in the fragment length. Hence it can be concluded that the ribosome will interact during initiation not only with the template segment directly covered by it, but also with the adjacent 5'-terminal region of RNA. Whether this interaction involves the completely formed 70S initiation complex or takes place at some intermediate stage of its formation remains to be seen. There are reasons to believe that such template-ribosome interaction extending beyond the ribosome binding site only provides additional stabilization of initiation complex. A similar stabilizing effect can be apparently exerted by the template segment to the right of the initiator codon, earlier characterized as being devoid of initiation determinants. Fragment (XVI) from the beginning of the replicase gene isolated from

Recognition of messenger RNA TABLE

I1

II

Fragments of phage RNA containing a complete ribosome binding site or part of it from one o f the genes used in reassociation experiments with the ribosomes in an E . c o l i initiation system in vitro. R17AP I

-20 -I0 -11 10 AUUCCUAGGAGGUUUGACCUAU.___G_GCGAGCUUUUAGU

R17CP I I

-20 -10 -11 10 AGAGCCCUCAACC§GGGUUUGAAGCAU___G.GGCUUCUAACUUU -40 -30 -20 -10 -II 10 AGGCAACGGCUCUCUAAAUAGAGCCCUCAACCGGAGUUUGAAGCAV.__GGGCUUCUAACUVU

III

-20 -I0 -11 10 AAACUUUGGGUCAAUUUGAUCAUGGCAAAAOUAGAG

qB CPIV -30

-20

-i0

-1]

10

UAAAGCGUUGAAACUUUGGGUCAAUUUGAUCAUGGCAAAAOUAGAG

V

-90 -80 -70 -6o AUCUUGAOACUACCUUUAGUUCGUUUAAACACGUUCUOGAUAGU-

vI

-50

-40

-30

-20

-10

-11

10

-AUCUUUUUAUUAACCCAACGCGUAAAGCGUUGAAACUUUGGGUCAAUUUGAUCAUGGCAAAAOUAGAG

-9o VII

-80

-70

-6o

AUCUUGAUACUACCUUUAGUUCGUUUAAACACGUUCUUGAUAGU-

-~o

-4o

-to

-~o

-io

-AUCUUUUUAUUAACCCAACGCGUAAAGCGUUGAAACUUUGGGU_CAAUUUGAUCAUG -10

-11

Relative

10

activity ~41

R17 RP VIII

AAACAUGAGGAUUACCCAU__.GGUCGAAGACAACAAAG

MS2 RP IX

-50 -40 -30 -20 -10 -11 10 20 30 40 ~0 cAAAc~ccGGcAUCuAcUAAUAGAcGCcGGccA~UcAAAcAUGAGGAUUAcccAU---5ucGAAGAcAAcAAAGAAGUuCAAcucUUUAuGUAUuGAUcUuccuCG1.0

X Xl

-50 -40 -30 -20 -10 -11 CAAACUCCGGCAUCUACUAAUAGACGCCGGCCAOUCAAACA~AGGAUUACCCAU__~GUCG

1.0

-50 -40 -30 -20 -I0 -11 CAAACUCCGGCAUCUACUAAUAGACGCCGGCCAUUCAAACAUGAGGAUUACCCAUG

1.0

-30

-20

-io

-11

0.7

AUAGACGCCGGCCAOUCAAACAUGAGGAUUACCCAU___~GUCG

Xll

-20 -10 -11 AOUCAAACAUGAGGAUUACCCAUGUCG

Xlll

-t0

-11

~0. I

AAACAUGAGGAUUACCCAUGUCG

XIV -50

f r RP XV

<0.1

-40

-39

-20

-10

-11

10

20

3.0

~AA~GGGAA~C~A~AAGAAA~G~GCCA~C~AA~AUGAGGAA~ACCC~GU~AAAA~CAACAAAGA~GUU~AA~CU~AUG -2o

XVl XVII XVIII XlX

-10

]0

20

39

1.0

CCAOUCCAACAUGAGGAAUACCC,~U___GGUCAAAAOCAACAAAGAAGUUCAACUCUUUAUG

0.4

-20 -I0 -11 10 CCAUUCCAACAUGAGGAAUACCCAUGUCAAAAUCAACAAAG

0.3

-20 -10 -11 10 CCAOUCCAACAUGAGGAAUACCCAUGUCAAAAOC

0.04

-20 -10 -11 CCAUUCCAACAUGAGGAAUACCCAU___GGUC

0.03

phage fr RNA, which is structurally identical over the ribosome binding site to the appropriate sequence in phage MS2 RNA [39, 40], was found capable of producing initiation complex, although with lower activity than the longer fragment (XV) [41]. Despite the presence of an almost equally sho~ 5'-terminal part, as in the inactive (XIII), fragment (XVI) owing to the nucleotide sequence following codon AUG exhibits its ribosome binding capacity. A shortening of the T-terminal part of fragment XVI leads to an BIOCHIMIE. 1984, 66, no I.

-11

abrupt drop in ribosome binding activity, when nucleotides belonging to the ribosome binding site are removed. For instance, fragment XVII still retains activity, for its 3"-terminal boundary coincides with that at the ribosome binding site, whereas fragments XVIII and XIX, under the same experimental conditions, are practically devoid of activity and therefore are analogous to phage MS2 RNA fragments XIII and XIV. Consequently, besides determinants of recognition specificity such as initiator codon and the

12

E.J. Gren

ribosome covered template segment on its left (i.e. the minimal initiation region), additional stabilization of initiation complex can be provided by the flanking region (i.e. additional interaction regions). The weaker the interaction between the ribosome and the main initiation determinants located in the minimal initiation region (to be discussed below), the more significant is the role played by this apparently nonspecific additional interaction and vice versa. Therefore, the sole active ribosome binding site under investigation is the phage RI7 A-protein gene region [31], participating in the strong interaction of the Shine-Dalgarno type (see below). However, a comparison of mRNA fragment activity must not neglect such an important discriminating factor as the spatial structure, especially when considering initiation regions of various genes differing in nucleotide sequence, or homologous fragments showing considerable variation as to their length. Finally, the above examples have been taken from phage RNA belonging mainly to phage MS2 and fr replicase gene, therefore it would be premature to speak of the universality of conclusions drawn with respect to the confines of initiation region on mRNA. There are other lines of evidence demonstrating the absence of initiation determinants beyond the ribosome binding site. In some deletion mutants of phage T4 rH B gene [42]; phage ~pX 174 F gene [43], phage ~. xis gene [44], E. coil l a c y gene [45], and Serratia mercescens trp L gene r',A1 ,t. ,~_.:~_ ~ q U ~ t l L . ~ i l l the re gi on j ust [L'WI, LI|U IIU~.,II~ULIU~ prior to the ribosome binding site is completely altered as well as its 5"-terminal nucleotides before the Shine-Dalgarno track. However, such crucial structural alterations had practically no effect on the corresponding gene activity or the deviations observed were only slight. In some instances, particular point mutations and deletions outside the ribosome binding site did lead to sig .ificant changes in translational activity of the appropriate mRNAs • phage T4 r l l B gene [42], E. coil lac z [46], arg E [47] genes, and phage cro gene [48]. However, in almost all cases, this could be regarded as a result of secondary structure alteration involving the ribosome binding site and the adjacent mRNA regions, e.g. as in cro gene [49]. .

.

.

.

.

Computerized processing of data concerning the primary structure of mRNA around initiator codon in many E. coil genes and those of its phages has revealed some common features shared by the nucleotide sequences at the initiation region (apart from the Shine-Dalgarno sequence BIOCHIMIE, 1984, 66, n° !.

and initiator codon). E.g., by comparing numerous structures, the most plausible model has been developed for the ,_'nitiation region [50] with prevalent A and U nucleotides on both sides of the initiator codon AUG (see table I). However, there is at present no experimental evidence indicating that any of the most plausible nucleotides found would be advantageous over others in a particular position. It is possible that the prevalence of A and U is merely associated with the necessity to avoid strong secondary structure in the initiation region, but G occurring only in the Shine-Daigarno sequence is perhaps due to the unambiguous positioning of the latter prior to the initiator AUG [5]. Another computational approach [51, 52] has also led to the identification of some structural features serving to distinguish true initiation regions of genes from other random DNA sequences with a similar structure. However, in this case, too, further investigation is needed along these lines. As for the coding part of mRNA following initiator codon, which exhibits some of the nucleotide sequence features, it seems more likely that it contains no structural determinants essential for initiation. Successful expression in E. coil of various eukaryotic genes with the hybrid ribosome binding sites being of bacterial origin prior to initiation codon also suggested a passive role for the 3'-terminal part of the ribosome binding site in initiation. Therefore, both genetic and biochemical findings suggest localization of initiation determinants within the ribosome binding site, or more exactly in its 5'-terminal part including the initiator codon (minimal initiation region). This does not signify, however, that the given region actually coincides with the complete initiation region and that the flanking regions play no part in initiation. The latter regions probably stabilize the initiation complex by nonspecifically interacting with the ribosome (regions of additional interaction). The positioning of the initiation region on mRNA is further complicated by the fact that the AUG or the short oligoribonucleotides containing the initiator codon as sole determinant are capable of binding to the ribosome to form a normal 70S initiation complex, although less efficiently than the natural mRNAs or their active fragments (see the next section). Furthermore, according to in vivo data, RNA transcript from phage L PRM promoter beginning exactly from phage ~. CI

Recognition o f messenger RNA repressor gene initiator codon AUG and containing no single nucleotide whatsoever on its left (see table I) is active in initiation and ~. repressor synthesis. However, the efficiency of translation is lower by an order of magnitude, as compared to the template with the normal left-hand part of initiation region and synthesized from a different PRE promoter [53, 54]. Unfortunately, there are no appropriate in vitro data available. Consequently, initiation is possible in principle, at the initiator codon on mRNA with a deleted left-hand part of the ribosome binding site.

3. Initiator Codons As can be seen from table I, the vast majority of prokaryotic genes use AUG as initiating codon, and only in some instances another codon, GUG, occurs. However, there are two more such codons • the genes for ribosomal protein $20 (rps T) [55l, NADH-dehydrogenase (ndh)[56] and, possibly also, lactose permease (lac a)[57] have UUG as initiator codon, whereas the gene for initiation factor IF3 (infC)[58] has even AUU. The ability of UUG triplet to act as initiator codon is also confirmed by the translational reinitiation data for E. coli iac i [59, 60] and phage T4 rIl B [61] genes. Amber mutations terminating the translation of consecutive mRNA regions ta~tal . . . . . . to the appro pfi ate nonsense codon are known to facilitate reinitiation of polypeptide synthesis from intragenic potential initiation sites after the termination codon. Among these reinitiation sites in lac i and rII B genes, apart from the conventional initiator codons AUG and GUG, UUG has been clearly involved. Irrespective of initiator codon structure, they all code for N-formylmethionine incorporation as N-terminal amino acid and therefore translation of initiator codon appears to have low specificity. In terms of anticoden CAU binding strength to the appropriate initiation codons, the first place is occupied by AUG, followed by GUG. Codons UUG and AUU show the weakest complementarity, provided by only two base-pairs. The role of complementarity in codon-anticodon interaction or, in a broader sense, in the interaction between fMet-tRNA and mRNA is inferred from the study of mutations affecting the initiator codon (table If). In most instances, AUG -* ACG, AUG -* AUA and similar mutations completely inactivate the gene or result BIOCHIMIE. 1984, 66, n° I.

13

in negligible activity (in the case c,f AUA) [44, 62-66]. Analogous inactivation of phage Q[~ coat protein gene was attained by the AUGG ~ AUAG mutation in the initiator codon. However, an additional mutation of the nucleotide next to the initiator codon AUAG ~ AUAA partly restored the ability of the gene te bind the ribosome [67]. This observation may be explained by assuming complementary base-pairing that takes place not only in the codon-anticodon region, but also at the adjacent nucleotides. In this case, the anticodon part UCAU has an additional base pair with a double mutant AUAA, as compared to AUAG. Such base-pairing of the nucleotide, just prior to acticodon with the corresponding nucleotide after the initiator codon, is confirmed by the occurrence of a mutation which does not affect the initiator codon (AUGG ~ AUGA in the same phage Q[~ coat protein gene). Mutant RNA (AUGA) binds more effectively to the ribosome within the initiation complex, as compared to the initial phage Q~ RNA (AUGG) [67]. A similar interpretation may be applied in the case of phage T4 rII B gene AUG ~ AUA mutation which is temperature-sensitive, i.e. is capable of initiating translation of the gene at a lower temperature [63, 64]. Additional complementary base-pairing on the other side of the codon-anticodon interaction (UUAUA with CAUAA) is conceivable in this case, partially making up for the mutational effect in the initiator codon. The involvement of nuc!eotides ajdacent to initiator codon in the formation of initiation complex is apparent from experiments modelling ribosomal binding to short AUG-containing oligoribonucleotides. It has been found that fMet-tRNA within initiation complex is actually capable of interacting with the nucleotides prior to the initiator codon, leading to enhanced stability of the complex. Tetranucleotide YAUG, especially UAUG, stimulates fMet-tRNA binding to the initiation complex much more efficiently than RAUG [68-71]. The same rule holds true for the synthesis of initiating dipeptides, where U A U G U U U appears to be a better template than A A U G U U U or AUGUUU [70]. Such recognition of the initiator codon context is apparently characteristic of the 30S initiation complex. This property of fMet-tRNA becomes lost upon 70S complex formation and is replaced by a conventional ~'iplet codon-anticodon interaction [69, 72]. The data concerning the recognition of the nucleotide next to initiator codon are not so unambiguous. According to some authors [72, 73],

14

E.J. Gren

AUGR are more readily recognized than AUGY (however, AUGA and AUGG do not differ), while other workers report [70] that the ribosome fails to distinguish between AUGU, AUGC, AUGA and AUGG. Equal template activity in the synthesis of initiating dipeptides has also been observed in the case of hexaribonucleotides [70]. As can be judged from the available data, the nature of the second and subsequent nucleotides on both sides of the initiator codon has practically no influence on the oligonucleotide activity in initiation [68, 701. The problem of relative activity in ribosomal binding by triplet AUG and longer AUG-containing oligoribonucleotides including mRNA is rather confusing. Addition of as many as 18 nucleotides 3" to AUG has hardly any effect on the ability of oligoribonucleotides to bind to the ribosome [68, 74-76]. Nucleotides 5' added to AUG exert a more pronounced effect, and, besides the above-mentioned influence of the purine or pyrimidine nucleotide preceding AUG, the very presence of the adjacent chain prior to AUG elicits a somewhat stimulatory action on ribosomal binding [77]; however, that needs to be confirmed. The data concerning the relative activity of the free trinucleotide AUG and the codon AUG contained in the native mRNA are rather contradictory. A comparison of the ability of pAUG triplet and phage Q~ RNA to form a 70S initiation complex has revealed that at equal molar concentration the RNA template is over t~an_fnltt

=.'t,,ia-lui'l.i

m,'~-a IIIUIV,,¢

,.~,-.,;~,,~ , l . . n - -

gvi~tllt,~

llliill

,1.~ lll~

...~--I--. I ' ' ? 0 1 lll~l~[

[101.

"T'LI_

1111~

11.__ llil~l

been also shown for MS2, RI7 RNAs and AUG [72, 79]. Other investigators failed :o report such a dramatic difference between ini.~i~ting triplets and mRNA [68, 74]. Despite the lack of systematic comparative studies on template activity of mRNA and short AUG-containing oligoribonucleotides, the general impression is that AUG and short AUG-containing oligoribonucleotides appear to be less active than the initiation regions in natural mRNAs, and that the high template activity cannot be attained by simple elongation of the nucleotide chain in either direction from the initiator codon. Whether the nature of the nucleotides located next to the initiator codon affects the process of initiation complex formation on natural mRNAs remains an open question. Using the data summarized in table I, the occurrence frequencies of the nucleotides next to the initiator codon have the following respective values -- prior : U-31 per cent, C-26 per cent, A-31 per cent, G-12 per cent; after : U-15 per cent, G-10 per cent, A-45 per cent, BIOCHIMIE. 1984, 66, n° 1.

G-30 per cent. Even in the case of initiator codon UUG, unfavourable in terms of complementarity with anticodon, it is surrounded by nucleotides far from optimal in view of facilitating interaction with fMet-tRNA. Initiator codon degeneration is amazingly high because of such, although rare, variants as UUG, AUU with weak classical type codon-anticodon interaction. It is possible that fMet-tRNA exhibits strong affinity for the P-site on the ribosome bound to mRNA initiation region, and that the codon-anticodon interaction is not so essential for initiation selectivity. The selectivity of initiator codon recognition is indicated by the structure of ribosome binding sites (table I). In some cases, despite the location close to the native initiator codon of other potential sites of initiation, such as AUG and G U G triplets, no initiation is observed. For example, mRNA for E. coli lactose operon repressor gene (lac i gene) has two GUG triplets in the same reading frame and an out-of-frame AUG triplet prior to the native initiator codon GUG, but none of them serves as initiation site. Mutation in the fifth codon A G G - - - A U G does not result in a new site of initiation either [60]. There is only one piece of evidence in the case of phage T7 0.3 gene indicating the use of AUG triplet close to the initiator codon in the same reading frame as initiation site, as a result of mutation of the true initiator codon AUG----ACG [621. Another example is provided by the weak initiation noted at the internal AUG, the third codon ~ , , . l g , l f f l l 3 L l g ; a , l l l llIL3111 I k l l ~ t ~ ' l ~ e 1 i11 LI/~l.l.O i- /-!, L J U l I l pnage . . . . fd gene 2 [80]. Discrimination between the multiple potential sites of initiation - - r a n d o m AUG and GUG triplets -- cannot possibly be accounted for in all cases by their protection against ribosome binding by mRNA macrostructure, especially when such triplet and the native initiator codon are contiguous. Therefore, there should exist some additional features distinguishing the true initiator codon which are encoded as a certain nucleotide sequence.

4. Shine-Dalgarno Seqence J. Shine and L. Dalgarno have been the first to propose a sound and tenable hypothesis postulating a certain nucleotide sequence in m R N A participating in initiation complex formation, apart from initiator codon [20, 81]. To date, this is the only hypothesis that has been, at

Recognition o f messenger R NA

least partially, experimentally substantiated. Having determined the nucleotide sequence of the 3'-terminal part of E. coil 16S ribosomal RNA as ...GAUCACCUCCUUAoH which predominantly contains pyrimidine bases, the authors hypothesized its interaction with a partially complementary mRNA region rich in purine nucleotides located prior to initiator codon. Such purine-rich regions occur in all mRNAs [82], their position being 3 to 13 nucleotides (mostly 5 to 9 nucleotides) to the left of initiator codon, 3 to 9 nucleotides being complementary (underlined in table I). This pufine-rich region in mRNA was later designated as the Shine-Dalgarno (SD) sequence and its interaction with the T-terminal part of 16S rRNA was denoted as the Shine-Dalgarno interaction. The earliest evidence in support of the ShineDalgarno hypothesis has been the direct demonstration of complementary interaction between the relatively short 3'-terminal s.c. colicinogenic 16S rRNA fragment and the ribosome binding site of phage Rl7 A-protein ger.e within a 70S initiation complex [83]. Similar supporting evidence has also been obtained for the initiation complex with phage ~. PR-transcript [84]. In both cases, the Shine-Dalgarno interaction involves 7 and 9 base pairs, respectively, facilitating complex fixation. However, the employed technique failed to establish such interaction in the case of poorer complementarity. The free E. coil 16S rRNA has also been found to bind oligonucleotide d-AAGGAGGT complementary to its T-end. 111%,, ,,.3 -I~III~I O

llUrl, J

lt%.l~l,/'l.

~Itlllllll tlll~; ..111J,J I I U U ~ U I I I d l

subunit remains exposed and interacts with the above oligonucleotide only in the presence of ribosomal protein S21 [85]. Somewhat unexpectedly, reconstituted 30S ribosomal subunits from 16S rRNA, the T-terminal nucleotides (around 160) of which were removed, appeared capable of binding fMet-tRNA on the poly (A,U,G) template [86]. This is only conceivable by assuming initiation complex formation on the template without the Shine-Dalgarno interaction. After the ShineDalgarno hypothesis, other types of interaction involving mRNA and 16S rRNA have been postulated, but they all lack experimental validation [87]. The Shine-Dalgarno interaction was also substantiated by.in vivo experiments. The study of mutants defective in translational initiation of phage T7 0.3 gene [62] revealed that mutation GAGGU (T7)-* GAAGU (CR35) in the ShineDalgarno sequence (table Ill) actually abolished its ability to participate in initiation (the middle nucleotide A drops out of complementarity to 16S BIOCHIMIE, 1984, 66, n° I.

15 TABLE II!

Mutations and o;her aherations in the translational initiation region ofsome mRNAs. QB CP ~ 6 7 ]

UUUGGGUCAAUUUGAUCAUGGCA

T7 0.3

GA C GCACGAGGUAALACAAGAUG

i62]

T4 r ll B [42 64]

AA G AA..AA GCCUAAUAAGGA~AUUAUGU--AC

yl. x i s ~ 4 4 ]

AUUGCGGAGACUuUGCGAUG

trp L ~ 6 5 j

UAAAAAGGGUAUCGACAAUG

tnp A i 8 9 ]

CAGGCAGGGAGGCCGAUAUG

c I I ~90]

G G 0 UAAGGAAAUI CUUACAUAUG

A, A6

A

lam B 188,139] ~GIb

1101]

A,

AU

G A A AUGACUCAGGAGAUAGAAUGAUGAUUACUCUGCG AGGAAACAG,GCAAUCCCCCAAAACAG~ACAGAAUG

SV 4 0 - t

~18~

AAUUUCASr~CAGGAAACAGAGC UU~GCAAAGAUG J,! ~ - -

sv 4 0 - t

39]

qbx 174 F ~b.3]

UUCACACAGGAAACAGCCAAGCUUUGCAAAGAUG jf &Lz GGGGCUUCGGCCCCUUACUUGAGGAUAAAUUAUG

sv 40-t

~105]

GCCUGCAUAAGGAGGUUUAAGCUUUGCAAAGAUG

s'~ 40-t

]06]

ACG#U GCCUGCAUAAGGAGGAGUU,U.GCA,AAGAUG

|ac z

~I04~

~x 174 H HGH

[I'3c~_

~99]

z cro sv 40-t

[48] ?07]

AUbCAG(_C ,~GA~AAUUCAUG ,% CUUPnGUGAGGUGAUUUAUG A ACAGC-~AACAGAAUO,CUAUG .5 ACAGGAAAcA'GCAUGUAC UAAGGAGGUUGUAUG

.~,~. . . .

.~ --

~GCAUUGGAAUUCCUCGAGGAAU~CCAAAGAUG

rRNA). Such a mutant, CR35, can synthesize the 0.3 gene protein in negligible amounts, its initiation probably taking place from a different AUG triplet-codon of the 6 'h amino acid. However, the CR35a-I revertant whith a mutation in the neighbouring nucleotide relative to the initial mutation in CR35 GAAGU (CR35)--~ GGAGU (CR35a-I) restores both the synthetic capacity and the site of initiation. In this event, the Cl~35a-I restores the Shine-Dalgarno sequence, although somewhat modified (GGAG) as compared to the initial GAGGU in the wild-type T7. A similar effect is exerted by mutations affecting the Shine-Daigarno sequence at the begin-

10

E.J. Gren

ning of phage T4 rll B [42] and E. coli lam B [88] gene (table liD. However, mutations destabilizing the Shine-Dalgarno interaction do not always lead to a suppressed or reduced translational activity of the corresponding mRNA. E.g., mutation GGAGG---~,~C,~GG occurring prior to tnp A gone in Tn3 transposone even results in enhanced gone expression [89]. This discrepancy is believed to result mainly from overlooking the fact that point mutations alter the spatial structure of initiator region. The spatial structure of mRNA at the initiator region as structural determinant of initiation will be dealt with in greater detail in the next section. In each case the steric factor should be taken into consideration when discussing any variations in the primary structure of initiator regions or even beyond them. For instance, nucleotide substitutions falling within the region between the Shine-Dalgarno sequence and the initiator codon decrease translational efficiency in some cases [42], stimulate it in others [89] or exert no effect at all [90] (table I11). Although the reasons may vary in each particular case, however, there seems to be a tendency for avoiding nucleotides C and G in this domain on the template, their introduction as a result of mutation generally leading to impaired translation. There is biochemical evidence in favour of the Shine-Dalgarno interaction. The synthetic oligoribonucleotide AGGACAUAUGp, resembling, to a certain extent, the ribosome binding site of phage QI~ A-protein gene, binds to the ribosome •~,,,~ ,t~ effectively as U C C U C A U A u t i p of equal length [77]. The capacity of oligodeoxyribonucleotide d-AATI'CTAGGATYrAATCATG to form initiation complex is comparable with that of the phage RI7 RNA and phage fd single-stranded DNA [79], Oddly, removal of the terminal ATG fails to affect the activitv of the residual oligonucleotide including the fMet-tRNA binding. Purine oligoribonucleotid,-s complementary to the 16S rRNA T-terminal l:yrimidine sequence can bind to the ribosome and 'Mock the formation of initiation complex with the native mRNA, but not with the short synthetic oligonucleotides [72, 78, 91-94]. For instance, oligoribonucleotide A.GAGGAGGUo, showing the highest complementarity of the Shine-Dalgarno type (AG" --16.9 kcal/mole) elicits a strong inh;.bitory effect on the ribosome binding by the phage Q[i RNA [78], while GGAAGGAGCoH (AG" - - l l . l kcal/mole) and GGAGAAGCoH (AG" - - 7 . 9 kcal/mole) are respectively less active. In the case of pAUG used as template, when initiation complex formation involves no ShineBIOCHIMIE, 1984, 66, n° i.

Dalgarno interaction, the above oligoribonucleotides do not exhibit inhibitory activity. The Shine-Daigarno interaction may proceed, to some extent, irrespective of the codon-anticodon interaction. The binding of E. coli ribosomes to the phage Q~ RNA template in the absence of fMet-tRNA yields small amounts of both 30S and 70S complexes, RNA :~gJons other than natural initiation regions being involved in ribosomal binding. The treatment of such complexes with pancreatic RNase resulted in three purine oligoribonucleotides AG.AGGAGGUp, GGAAGGAGCp and AGGG. GGUp, resistant to the enzyme,which were definitely complementary to the 3'-end of 16S rRNA ...ACCUCUUAca [95]. The first two fragments, as noted before, may compete with Q[i RNA for ribosome binding [78]. Interaction of AGAGGAGGUp with the 16S RNA T-terminal fragment within the ribosomal complex has been clearly demonstrated [95]. Consequently, in the absence of fMet-tRNA the ribosome randomly selects accessible mRNA regions with a sufficiently long Shine-Dalgarno sequence. A similar picture is obtained during the binding of B. stearothermophilus ribosomes with the template. The 3'-terminal structure of the bacterial 16S rRNA ...GAUCACCUCCUUUCUAoH is practically identical to that of E. coli, except for the three underlined additional nucleotides at the extreme 3'-end, suggesting a similar Shine-Dalgamo interaction in both cases [96]. The B. stearothermophilus ribosomes, under optimal binding conditions at 65oC, failed to bind to the phage Q[I RNA natural initiation regions even in the presence of fMet-tRNA, but formed fairly stable complexes with the template regions devoid of AUG. This was confirmed by the isolation of CUGAAAGGGGAGAUUACUCG and G G A A G G A G C with a distinct Shine-Dalgarno sequence from the complex. Using phage RI7 RNA as template, the thermophile ribosomes are only capable of binding to the A-protein gone initiation region comprising an extended ShineDalgarno sequence [96,97]. When the temperature of incubation was lowered to 49oC, less stable complexes, characterized by a weaker Shine-Dalgarno interaction, were formed. As a result, ribosomes of B. stearothermophilus ~-t 49oC, apart from binding to the phage R17 A-protein gone initiation region, also interacted with the replicase gene initiation region, but with a lower activity. In the case of phage QI~ RNA, both the replicase and coat protein gene initiation regions have been found active in ribosomal binding at 49oC [96].

Recognition of messenger RNA

The temperature dependence of initiation complex formation by B. stearothermophilus ribosomes demonstrates the importance of ShineDalgarno interaction in the formation of initiation complex, though the situation is clearly complicated by the temperature effects on the template macrostructure itself. However, the above observations clearly indicate a relationship between the stability of initiation complex and that of the Shine-Dalgarno interaction. The firmer this interaction, the less imvortant is the role of codon-anticodon and other contacts. Indeed, initiation complex formation involving replicase and coat protein genes~on the phage Rl7 RNA template is much more effectively stimulated by fMet-tRNA and initiation factors, as compared to the A-protein gene characterized by a more stable Shine-Dalgarno interaction [31,98]. However, this does not imply the existence of a relationship between the Shine-Daigarno interaction energy and efficiency of initiation on various mRNAs; it is far from being the case. For example, among the three genes of the native phage Q[~ RNA, the highest initiation efficiency is displayed by the coat protein gene possessing the shortest Shine-Dalgarno sequence. This discrepancy is mainly explained by the fact that the role of template macrostructure essential for initiation has not been taken into consideration (see the next section). This applies primarily to the shielding effect of the mRNA macrostructure, although the role of protein-RNA interaction in the stabilization of initiation complex involving initiation factors and ribosomal proteins, and apparently dependent on the structural features of the template, cannot be neglected either. Furthermore, one cannot draw a parallel between initiation complex stability and initiation efficiency, because of the lack of conclusive evidence. To exemplify the point, similar ribosome binding activity can be adduced for the wild-type, phage T7 0.3 gene mRNA and its mutants CRI7, CR35a-I, their initiation efficiency being drastically different [62]. Therefore, the most suitable efficiency criterion for translational initiation would be template directed binding of fMettRNA to the ribosome and the synthesis of initiating dipeptides, as compared to the mere binding of the template to the ribosome yielding initiation complex. Finally, during initiation the ribosome might possibly participate in complementary coupling not only with the Shine-Dalgarno sequence, but also with a more protracted template region including the mosaic type of interaction when BIOCHIMIE. 1984, 66, n° l.

17

gaps and loops are left on the interaction region. It is fairly difficult to assess the destabilizing effect of such gaps and loops on the complementary binding of RNAs especially in protein surroundings within the ribosomes. It is not surprising that using a given approach no direct correlation has been found between the stability of the conventional Shine-Dalgarno interaction and the efficiency of initiation complex formation. However, such speculations are rather arbitrary and lack experimental basis at present. As follows from table I, the Shine-Dalgarno sequence is located prior to the initiator codon at different spacings. This fact still requires interpretation; there are no clear notions either as to the optimal location of the Shine-Dalgarno sequences. In vivo studies on the expression of hybrid and reconstituted genes point to a possibility of variation in the distance between the ShineDalgarno sequence and the initiator codon without essentially affecting the synthesis of the appropriate protein. In E. coli lactose operon, the initiator codon of the first [~-galactosidase (lac z) gene is spaced 7 nucleotides appart from the Shine-Dalgarno sequence AGGA. By inserting a synthetic human growth hormone gene at the lactose operon promoter, the distance between the Shine-Dalgarno sequence and the initiator codon as well as the nucleotide sequence between them remaining the same, normal expression of the gene can be achieved. Moreover, addition of four extra nucleotides to the sequence even leads to a somewhat enhanced efficiency of the growth hormone synthesis [99]. Using a recombinant DNA technique, the lactose operon promoter together with the lac z gene Shine-Dalgarno sequence (AGGA) and the five nucleotides following it have been added to the structural part of different genes, the initiating AUG being placed at a varying distance from the Shine-Dalgarno sequence (table III). Effective synthesis of SV 40 viral t-antigen was elicited when the distance was equal to 8 and 9 nucleotides, but it dropped by two-fold at I I nucleotides, at the extremely long distance of 17 and more nucleotides [18, 19] the synthesis was decreased by more than ten-fold. Normal expression of the phage L CI gene was achieved with a 8 nucleotide spacing [100]. Appreciable rabbit [~globin synthesis was noted when the lac z gene Shine-Dalgarno sequence was located 8 to l0 nucleotides apart from AUG, but at 27 nucleotides and more a marked drop was obtained [101].

18

E.J. Gren

A weak, but still noticeable mRNA translation of cloned genes with the lat z gene Shine-Dalgarno sequence removed far away from the gene start is provided without its participation in initiation, or without the Shine-Dalgarno interaction at all, or by randomly using the partially complementary purine nucleotides prior to the AUG codon. This type of translation initiation in the absence of the natural Shine-Dalgarno sequences is not unfrequently observed during gene cloning, the mouse dihydrofolate reductase presenting a better studied case [102, 103]. Oligo(G) linkers prior to the cloned gene may also serve as synthetic Shine-Dalgarno sequences. Expression of some genes artificially fused with chemically synthesized linkers including a Shine-Dalgarno sequence has been studied. An equally effective lac z gene expression was attained with 8 to 9 nucleotides between the ShineDalgarno sequence and the AUG codon [104]. Active synthesis of the SV-40 viral t-antigen was observed when sequence UAAGGAGGU was I I and 12 nucleotides from the gene start, but suddenly dropped by more than an order of magnitude when the distance was increased by l or 2 nucleotides [105, 106] (table III). Systematic variation in the distance between the Shine-Dalgarno sequence and the AUG codon revealed a poor correlation with the efficiency of synthesis of the SV-40 t-antigen [107, 108] (table Ill). The amount of synthesized t-antigen varied over a wide range (by more than ~ - t u ~ u ) not omy as a function ol olstance between the initiatory AUG and a rather weak Shine-Dalgarno sequence GGA (AGGA in some cases), but also depending on the primary structure of the ribosome binding site itself and especially of the preceding region. These data could be explained only by analyzing the secondary structure models for the 5'-terminal part of the mRNA covering several hundred nucleotides (see below). A detailed analysis helped to unravel even the complicated pattern of events observed with the phage ~. cro gene expression in various recombinant contructions [48, 49]. Among the two native sites for the ohage fd gene 2 translation initiation the highest activity is shown by the AUG removed from the ShineDalgarno sequence by 4 nucleotides. Another AUG, 13 nucleotides from the same Shine-Dalgarno sequence, is markedly less active in i=~itiation (10 per cent of the first AUG activity) [80]. During the SV 40 t-antigen synthesis in recombinant clones under the phage ~. PL-promoter control, initiation from another AUG codon apart BIOCHIMIE. 1984, 66, n° !.

from the main initiation point at the natural initiator codon has been discovered [107]. However, it was found that among the two closest intrinsic AUG triplets in the reading frame of the gene, only the second one is active in initiation. Like the natural initiator codon in mRNA, it is exposed to the ribosome. Several deletions have been described as affecting the distance between the Shine-Dalgarno sequence and the AUG codon [42, 109], however, their effect on the expression of the corresponding gene still awaits a satisfactory explanation. Despite the ambiguity noted above and the difficulties involved in the interpretation of the practical gene engineering data, high levels of direct expression of cloned genes have been achieved by preserving reasonable distances between the natural bacterial Shine-Dalgarno sequence and the beginning of eukaryotic genes. Thus, the iac-promoter regulatory region has provided the synthesis of fibroblast and leukocyte interferon [110-114], and src-protein [156] in E coli. Effective synthesis of fibroblast and leukocyte interferon [l I l, 115-118], serum albumin [i 19] and hepatitis B viral core-antigen [120] has been achieved by utilizing the E. coli trp-operon regulatory region. Practically involved was the distance between the gene start and the natural or synthetic Shine-Dalgarno sequence about 7-9 nucleotides long.

5. Macromolecular Structure The importance of the mRNA macromolecular structure in translation has been first demonstrated using RNA-containing phages. Only one of the t.hree phage genes in the native RNA, that for the coat protein, is exposed to ribosome bindin~g. The other two genes, those for replicase and A-protein, become accessible to the ribosome only after limited phage RNA fragmentation [25, 26, 28-30] or partial unfolding of its macrostructure by way of chemical modification with formaldehyde [121,122]. The gene for A-protein somewhat removed from the 5'-end of the template can be expressed only as part of relatively short (several hundred nucleotides) 5"-terminal RNA fragments, their elongation towards the 3"-end leading to the shielding of the initiation region [28, 123]. This is functionally important, because the A-protein gene is expressed only on short, so-called nascent, chains within the phage replication complex. The A-protein gene is inactive in the full-length native phage RNA. This

Recognition of messenger RNA

type of regulation can be achieved by a specific interaction between the A-protein initiation region and some region on the phage RNA removed from it towards the 3'-end [124], although the precise position of the interacting regions is unknown. The RNA phage binding by the B. stearothermophilus ribosomes yielding initiation complex takes place at a high temperature (65oC), when RNA macrostructure, including its part shielding the A-protein initiation region, is partially destroyed, the latter efficiently binding to the ribosome. At 37oC this interaction is decreased by 20-fold [122]. Functionally different (but apparently structurally similar) macrostructure shielding is observed during temporary closure of the rep!icase gene initiation region of phage MS2 [125] and other related phages. In this case, partial unfolding of macrostructure removed the shielding effect [121,122]. The same is true for the phage gene four, i.e. the gene for L-protein [126, 127, 128]. Besides RNA phages, other examples are available of translational regulation by means of mRNA macrostructure elements involving the initiation region. An interesting example of the so-called translational attenuation has been proposed for the B. subtilis erm C gene. The gene for the short leader peptide is located prior to the erm C gene. In the template common for the two genes, the erm C gene start is shielded with participation of the leader peptide coding sequence. "l'he erm C gene initiation region becomes exposed to the ribosome only after translation of the latter sequence [129]. The long E. coil gal-operon transcript from the phage lambda PL-promoter shields the proximal g a l E gene initiation region by incorporating it into a stable helix, whereas transcription from the gai-operon promoter does not prevent ribosomes from easy initiation at the gal E gene [130]. The primary transcript from the phage T7 gene 0.3 is inactive with respect to translational initiation, this effect being abolished during the template processing by the removal of the noncoding 5'-terminal part with RNase III [131]. In this case too one deals with the shielding effect of RNA macrostructure. The evidence presented and other similar cases clearly reflect the shielding role of mRNA macrostructure in translational initiation regulation of certain genes, but we are still unaware of the concrete mechanism underlying the shielding effect. Even the generally accepted principle of BIOCHIMIE. 1984, 66, n° I.

19

initiation region inactivation by way of its binding to another complementary region on the message has been proved rather by circumstantial evidence than by direct experimentation. Except for some rare cases, practically all the models of proposed interaction are based either on maximal basepairing calculation or, at best, on initiation region accessibility to RNase attack or electrophoresis of interacting oligonuc!eotides following partial RNase digestion of mRNA [125, 132-134]. This results from many unsurmontable obstacles lying in the way to the determination of precise secondary structure of RNA and fragments thereof. It would not be tight to assume that any secondary structure comprising an initiation region with initiator codon and a Shine-Dalgarno sequence precludes initiation, the completely exposed or partly buried initiation domains being active. The only presently known initiation region located on a short mRNA fragment whose supposed structure has been more or less proved by physical methods is the phage R17 and MS2 replicase gene initiation region within the 59-nu-

(b)

(o)

cUAA A U -•AU -4oC.G 3o U-A-

uAc.TC

"~r_t. UA -C

u

G.C

[U-.~ °I &olJ

E.~

E.F=

-5o uC°Gc -2o

5'...CAAAC

~,~.c-,

CAUUCA

AA'UCG 5' ...

FIG. 1. - - The most probable secondart." structure model of phage MS2 RNA fragment prior to the beginning of the replicase gene developed on the basis o f physico-chemical data and maxinwi base-pairing calculations [33, 40]. Base substitutions in the appropriate phage RI7 RNA fragment are indicated [135, 136]. Initiation codon is framed, one of the Shine-Dalgarno sequence variants is underlined.

cleotide fragment [135-137] (fig. I) or the shorter polynucleotide only coveting the hairpin (b) of the 59-nucleotide fragment [138]. The structurally similar phage f r R N A segments can also be assigned to them [40]. Hairpin (b) whose helical part contains both a Shine-Dalgarno sequence and an initiator codon melts in a high ionic strength buffer at 61oC [135]. Nonetheless, the fragment is highly effective with respect to the initiation complex formation, its activity being comparable with that of the native phage RNA [33]. It is possible that the resulting open form of

20

E.J. Gren

the hairpin (b) region in solution at 37oC, being in equilibrium with the basic helical structure, represents the active form involved in the interaction with the ribosome. However, it would be justifiable to assume that the ribosome can easily unwind the helix, a process which cannot take place during interaction in the case of fairly strong complementary complexes. Using the computational models of the secondary structure of several initiation domains and the adjoining mRNA regions, some authors have attempted to treat translational initiation data available for different genes in relation to their hypothetical secondary structure. Thus, reinitiation points in the E. coli lac i gene have been analyzed rather successfully with respect to their accessibility to the ribosome in nontranslating mRNA and during disruption of local secondary structure elements in the message as a result of selective translation [60]. This has enabled to somewhat clarify the intricate sequence of events involved in the phage ~. cro gene translation in several plasmid clones differing in the nontranslaring region prior to the AUG codon [48, 49]. A similar approach has been applied to study the expression of cloned SV 40 t-antigen gene [107]. Secondary structure computations conducted for rather lengthy (several hundred nucleotides long) sequences around the initiation codon have revealed a number of clones whose initiator codon for cro and t-antigen gene, but not necessarily the Shine-Dalgarnn _~eq.~..-,% i~ i . )h . . . . k~. lical part and must be accessible to the ribosome. These variants have been found to engage actively in expression, unlike the other ones whose initiator codon is located in the helical part of the structure [49, 107]. _

--

.

.

.

.

.

.

.

.

.

.

v v

i~.J

lag

L I I ~

[140]. A comparison of secondary structure models in the DNA-containing phage and bacterial m R N A regions around translational initiation sites has revealed some similar elements indicating that in the fairy structural initiation regions the Shine-Dalgarno sequence must be located in the nonhelical loop part of the hairpin, while the presence of the initiator codon in it is not so obligatory. According to these calculations the loop regions may contain from 5 to 61 nucleotides, whereas the helical part comprises from 7 to 14 base pairs (AG ranging from - - l to - - 3 8 kcal/mole) [141]. However, this statement cannot be extended to the vast majority of the RNA phage genes and E. coli genes for the ribosomal proteins [142]. The above mentioned analysis of plasmid variants of the phage ~.cro gene [49] and of the SV40 t-antigen gene [107] indicates that the location of initiation codon, but not the Shine-Dalgarno sentlence

in

th@

i,~,~

~.,~,~;~..

;. . . . . .

.:_t

e__

l l U I I I I ~ "

The effect of some point mutations occurring in the initiation region or outside it on the translation of certain genes may also be interpreted in terms of secondary structure models. A most peculiar case is presented by two transversions in the E. coli lam B gene -- 701 and 708 -lying on the boundary of the ribosome binding site. Mutation 701 ( U - * G ) affects the 12th nucleotide before the initiator codon (2nd nucleotide prior to the Shine-Dalgarno sequence); mutation 708 (C--~ A) affects the first nucleotide of the 5th codon in the gene [88, 139]. Each of the two mutations reduces the translational activity of the lain B gene by approximately ten-fold, but placed together they restore the gene activity. The seeming paradox can be readily explained using the secondary structure models for this segment BIOCHIMIE, 1984, 66, no !.

of the template. Each of the two mutations enhances stability of the helix comprising also the Shine-Dalgarno sequence, whereas the double mutation destabilizes the helix similarly to the wild type. Sometimes mutations located far from the gene start can affect its translational activity, such as those detected on the 22nd and 26th nucleotide prior to the E. coil arg E gene [47] or in the phage T7 0.3 gene itself (in the coding part 70 nucleotides from the beginning of the gene)

expression. An interesting hypothesis has been put forward postulating that certain elements of secondary structure can even stimulate initiation [143]. The possibility of hairpin formation has been hypothesized in various mRNAs to the left from the initiator codon, the initiator codon as well as the Shine-Dalgarno sequence being located within these structures in the nonhelical region. The presence of a hairpin in the initiation region or the possibility of its formation during the recognition of the template by the ribosome may play a guiding role in initiation complex formation. This hypothesis, however, needs experimental verification. Consequently, the discriminating role of mRNA macrostructure in gene expression has been generally accepted and is well-provided with experimental evidence, although the fine structure of such interacting mRNA regions as well as the spatial structure elements in the active initiation sites are practically unstudied.

Recognition o f messenger RNA

The mRNA macrostructure also shields, apart from ~he native initiation regions, multiple sites of false initiation. Disruption of phage f2 RNA macrostructure by complete chemical modification of cytosine residues by methoxyamine has led to the appearance of multiple ribosome binding sites, the incorporation of fMet-tRN,~ into initiation comp!ex being increas,~.d by 50-fold, as compared to the native t"2_RNA template [144-146]. According to some investigators, such a sharp rise in the number of ribosome binding sites on the template with unfolded macrostructure has put to considerable doubt the role of the Shine-Dalgarno interaction during initiation complex formation. In their opinion [147-149], any initiator codon, whose spatial location is accessible to binding by the ribosome, can direct initiation, the template macrostructure being the only selective factor chosing true initiation codons among the many spurious ones. By using the statistical copolymers-polyribonucleotides, they discovered that the most active in ribosome binding were the low-structured polymers of poly(G,U) type, while the poly(G,U,C,A) showed a very small activity, especially when the G:C and A:U ratios were approaching one. The natural mRNAs are believed to possess no advantage in terms of initiation efficiency when compared to the synthetic polyribonucleotides. In general, all mRNAs bear resemblance to poly(G,U,C,A) with multiple potential initiation codons buried in the macrostructure, only the true initiator codons remaining exposed. Although the experimental evidence presented appears rather convincing it is at odds with some earlier mentioned facts indicating the importance of the Shine-Dalgarno interaction both in vivo and in vitro.

6. Termination Codons It does not take a close scrutiny of the ribosome binding sites (table I) to note that practically all of them contain one or more termination codons, such as UAA, UAG or UGA, in or out of the reading frame of the gene, located in the coding part or prior to the initiator codon. This phenomenon may be fortuitous, since the appearance of a termination codon in any reading frame is possible after every 7 codons. However, some investigators [68, 150] also involve in initiation the termination codons falling within the mRNA region prior to the initiator codon, conferring to them the role of ribosome conditioning for BIOCHIMIE, 1984, 66, n ° I.

21

the initiation, which still needs experimental verification. The 16S rRNA T-terminal nucleotides can actually participate in the complementary interaction with two termination codons -- UAA and UGA -- hence they are probably expected to take part within the ribosome in the recognition of termination codons also during initiation [21,81]. The termination codon may constitute part of the Shine-Dalgarno sequence. It is interesting to note that the termination codon closest to the initiator codon is mostly UAA or UGA, but not UAG. This has led to conclude that only two codons - - U A A and U G A are involved in initiation [ 151 ]. The issue of the possible involvement of termination codons in the formation of initiation signal on mRNA is related to the problem of translation reinitation for a new gene following the termination of the previous one. When the intragenic area is sufficiently long one can expect independent termination and initiation events, except for the cases when translation of the distal part of the preceding gene can affect the spatial structure of the initiation region of the following gene. However, in the cases where the termination codon of the preceding gene is closely located to the initiation codon of the following gene within the ribosome binding site, or even shows partial overlapping, one can hardly conceive that the two events are independent. Such cases are not so rare and they are known not only for DNA-containing phage, but also for bacterial genes belonging to the same operon; e.g. in the trp-operon; moreover, the initiator codon can be located even to the left from the termination codon. The functional implications of the close spacing of the terminal and starting points of two genes are not clear, although in such cases one can at least imagine the effect of the translation rate of the preceding gene on the initiation efficiency of the following one. It is generally believed that the ribosome after termination leaves the template and is ready for reinitiation at a new initiation site. However, in the case of closely located or even virtually overlapping termination and initiation points, one can suggest that the ribosome after termination does not leave the template or being dissociated from it creates a locally high concentration of ribosomes at the new initiation site. In any case, after approaching a new initiation region, the ribosome forms an initiation complex with it [152, 153], increasing the translational initiation rate of the following gene. In the absence of releasing factor in an in vitro system the ribosome is capable of reinitiation at

22

E.J. Gren

the amino acid codon following the nonsense codon requiring neither the initiator codon, nor the Shine-Daigarno sequence [154]. However, in vivo the presence of an initiator codon after the nonsense codon within the gene has been established, but not that of the Shine-Daigarno sequence [60, 61]. Reinitiation is not an artefact occurring in genes affected by nonsense mutations. A special type of reinitiation can be found during the phage MS2 L-protein gene translation. Although there are nucleotides resembling a Shine-Dalgarno sequence prior to the L-protein gene, they can be deleted without any significant consequences, since the gene translation is not autonomous, but proceeds in conjunction with the out-of-frame translation of the preceding part of the coat protein gene. According to the proposed model [128], during the coat protein gene translation an insignificant part of the ribosome changes the reading frame of the gene and comes across the termination codons in both misreading frames. Such termination codons are located close to the L-protein gene start, and the ribosomes after termination reinitiate at the L-protein gene AUC codon. If these two contiguous termination codons are deleted, or the template is translated in the reading frame for the coat protein gene, initiation of L-protein synthesis fails to take place. Actually, the reinitiation phenomenon should not be necessarily regarded as a special type of tandem translation of two adjacent mRNA regions or genes, separated by a termination codon, ~,: . . . . . . . ,.+ , , , . , , . , + , . , , , , + . , u , u u t ,,+ dissociation from the template. Reinitiation may occur after the usual mechanism of translational initiation and is invariably accompanied by the unfolding for the translating ribosome of macrostructure shielding the new initiation site. Whatever the true mechanism of reinitiation, a relationship is clearly obvious between the gal K gene translational initiation and the location of termination site in the preceding gal Tgene [155]. The closer the location of the termination codon to that of the initiator one, the more effective is the gal K gene expression. With early termination before galK gene or during out-of-frame translation with termination within the galK gene, when the termination point is detached from the gal K initiation region, the gal K gene expression is markedly reduced. The close tandem spacing of genes within the single operon, as in the trpoperon or in the ribosomal protein operons, apparently proviaes a stoichiometric and effective synthesis of such proteins within the operon, and probably fulfills a regulatory function.

BIOCHIMIE. 1984, 66, no I.

7. Conclusion Despite the fairly intensive investigation of the structural aspects involved in the mRNA recognition by the ribosome during translational initiation in prokaryotes, the general picture remains rather obscure. There is a lack of consensus even with respect to the general principles involved (e.g. the Shine-Dalgarno hypothesis), as to their real contribution to the initiation process. This may be explained by the multivalent nature of initiation determinants, when the integral signal formed, guiding the ribosomal binding to the initiation region at the beginning of the gene, consists of several components, each of them having a different directing force in each particular case. Structurally different mRNA regions appear to have interacted with the ribosome during the evolution of the initiation regions and the ribosome has learnt to distinguish them sometimes by fairly different features. Therefore, it seems unwise to reduce the entire problem of RNA template recognition by the ribosome during initiation to only the mere identification of common universal structural determinants among the multiple initiation regions. It appears more justified to study the impact of any given structural element on particular steps of initiation or on the process in general, especially in quantitative terms. Therefore it cannot be ruled out that in view of the muitivalent nature of initiation signal, the absence or weak functioning of some elements may be compensated by a more active involvement of other elements. Surely, such a problem is much more difficult to tackle. Among the most useful approaches to the solution of this problem, on should mention the study of initiation with middle-size synthetic oligorib0nucleotides, containing both the structurally and positionally variable Shine-Dalgarno and initiator codon sequence as well as strictly fixed elements of secondary structure. Their detailed investigation during the first cycles of elongation may provide answers to many so far hypothetical conclusions. Another advantageous approach may be the study of initiation in vivo using mutant and hybrid initiation regions at required positions which are conceivable only by applying the recombinant DNA and site-directed mutagenesis t:.~chniques. An all-round investigation of transcription and translation in a given gene including mRNA stability data can considerably add to the in vitro studies with model oligoribonucleotides.

Recognition o f messenger RNA

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27. Gupta, S.L., Chen, J., Schaefer, L., Lengyel, P. & Weissman, S.M. (1970) Biochem. Biophys. Rex. Communs, 39, 883-888. 28. Staples, D.H., Hindley, J., Biileter, M.A. & Weissmann, C. (1971) Nature New Biol., 234, 202-204. 29. Staples, D.H. & Hindley, J. (1971) Nature New Biol., 234, 21 ! -212. 30. Steitz, J.A. (1972) Nature New Biol., 236, 71-75. 31. Steitz, J.A. (1973) Proc. Nat. Acad Sci. USA, 70, 2605-2609. 32. Berzin, V.M., Borisova, G.P., Gribanov, V.A., Rosenthal, G.F., Cielens, I.E., Jansone, I.V. & Gren, E.J. (1976) Dokl. Akad. Nauk SSSR (in Russian), 229, 741-744. 33. Berzin, V., Borisova, G.P., Cielens, I., Gribanov, V.A., Jansone, I.,, Rosenthal, G. & Gren, E.J. (1978) J. Mol. Biol., 119, 101-131. 34. Adams, J.M., Cot3,, S. & Spahr, P.F. (1972) Europe. J. Biochem., 29, 469-479. 35. Porter, A.G. & Hindley, J. (1973) FEBS Letters. 33, 339-342. 36. Weber, H., Billeter, M.A., Kahane, S., Weissmann, C., Hindley, J. & Porter, A. (1972) Nature New Biol., 237, 166-170. 37. Jansone, I., Berzin, V., Gribanov, V. & Gren, E.J. (1979) Nucl. Acids Res.. 6, 1747-1760. 38. Borisova, G.P., Volkova, T.M., Berzin, V., Rosenthai, G. & Gren, E.J. (1979) Nucl. Acids Res.. 6, 1761-1774. 39. Berzin, V.M., Gribanov, V.A., Cielens, I.E., Jansone, I.V. & Gren, E.J. (1981) Bioorganicheskaya khimia (in Russian) 7, 306-308. 40. Cielens, I.E., Jansone, I.V., Gribanov, V.A., Vishnevsky, Yu. I., Berzin, V.M. & Gren, E.J. (1982) Molekularnaya biologia (in Russian), 16, ! 109-1 ! 15. 41. Berzin, V., Cielens I., Jansone, 1. & Gren E.J. (1982) Nucl. Acids Res., I0, 7763-7775. 42. Singer, B.S., Gold, L., Shinedling, S.T., Coikitt, M., Hunter, L.R., Pribnow, D. & Nelson, M.A. (1981) J. M6,1. Biol., 149, 405-432. 43. Miiller, U.R. & Wells, R.D. (1980) J. Mol. Biol., 141, 25-41. 44. Abraham, J., Mascarenhas, D., Fischer, IL, Benedik, M., Campbell, A. & Echols, H. (1980) Proc. Nat. Acad. Sci. USA, 77, 2477-248 I. 45. Mieschendahl, M. B0chel, D., Bocklage H. & Miiller-Hill B. (1981) Proc. Nat. Acad. Sci. USA. 78, 7652-7656. 46. Cannistraro, V.J. & Kennell, D. (1979) Nature, 277, 407-409. 47. Boyen, A., Piette, .~., Cunin, R. & Glansddorff N. (1982) J. Mol. Biol., 162, 7 i 5-720. 48. Roberts, T.M., Kacich, R. & Ptashne, M. (1979) Proc. Nat. Acad. Sci. USA, 76, 760-764. 49. Iserentant, D. & Fiers W. (1980) Gene, 9, i-12. 50. Scherer, G.F.E., Walkinshaw, M.D., Arnott, S. & Morre, D.J. (1980) Nucl. Acids Res., 8, 3895-3905. 51. Stormo, G.D., Schneider, T.D. & Gold, L.M. (1982) Nucl. Acids Res.. 10, 2971-2996.

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