Invivo maturation of an undermodified Escherichiacoli leucine transfer RNA

Vol. 73, No. 2, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

--IN VIVO MATURATION OF AN UNDERMODIFIED ESCHERICHIA COLI LEUCINE TRANSFER RNA Geoffrey

R. Kitchingman*

and Maurille

J.

+

Foumier

Department of Biochemistry University of Massachusetts Amherst, Massachusetts 01002 Received

September

lo,1976

SUMMARY: Two chromatographically unique leucine tRNAs which accumulate during leucine starvation of relaxed control E. coli have been shownLg; be undermodified forms of the major, normal isoacceptor species, tRNA . Results from 32P label-chase experiments demonstrate that the undermo 3 ified species, deficient in specific dihydrouridine and Egzudouridine residues, can be converted to chromatographically normal tRNAI but, only slowly. The slow rate of conversion suggests that the undermodified isoacceptors are not normal intermediates in the biosynthetic process and provides additional support to the view that certain of the post-transcriptional modification reactions occur in a sequential manner. It

INTRODUCTION:

is well

of tRNA can occur infection,

under

cellular

known,

but

is

believed

modification

of the

Modification

deficiency in the spectra

E. coli

starved in the

adenosine

(9)

also

of rel-

and

a precursor

Changes

the biochemical

has been

shown

of methylated

(10)

cells

starved

0 I976 by Academic Press, Inc. of reproduction in any form reserved.

314

is not

of amino

for

amino

acids

are

threonylcarbamoylmethionine

(4abu3U-ref.

been observed acids

the

(rel-)

respectively;

uridine have also

basis control

(8),

to 3-(3-amino-3-carboxypropyl)

bacterial

observed

These

nucleosides

patterns

conditions

of tRNA gene expression.

or cysteine.

nucleosides

viral

of post-transcriptional

tRNAs from relaxed

Laboratory of Molecular *Present address: Institutes of Health, Bethesda, Maryland should be directed.

Copyright All rights

the changes

to be the biochemical

threonine

in tRNA isoacceptor

tRNAs from

modulation

of isoacceptor

sulfur-containing

for

patterns

including:

in culturing

to the extent

than

the isoacceptor

conditions

basis

to be related

synthesis

in

and alterations

of methionine,

involved

changes

of metabolic

tRNA rather

changes

is

a variety

that

differentiation

In most cases,

(1-7).

documented

not

for

known

Genetics, NICHHD Bldg. 20014. +To whom requests

11). a number to be

6, National for reprints

BIOCHEMICAL

Vol. 73, No. 2, I 976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Figure 1. Effect of leucine starvation on the leucine tRNA from relaxed control E. coli. Samples of tRNA from leucine-starved (6 hrs) and nonstarved (mid-log) cultures of E. coli CP79 (arg-, his-, leu-, thr-, thi-, rel-) were aminoacylated with 3H-and I4C-1 eucine respectively, mixed and analyzed by RPC-5 chromatography. Fractions were precipitated with 5% trichloroacetic acid and the insoluble material collected on Millipore glass fibre filters. The 3H- and 14C-activity profiles were determined by liquid scintillation spectrometry and normalized to the same total activity. (X X) tRNALeu from nonstarved cells (NS), [14C] leucine; (o - - - l ) tRNALeU from leucinestarved cells (-leu), [3H] leucine.

involved

in tRNA base modification

inhibitors

of protein

on the

isoacceptor

in Fig. the

1.

cells

species

tRNA Leu isoacceptor

unique (fractions

148-155).

homogeneous

major

can be partially cation

cells

The major

unique

five

a rel-

or six contains

of tRNA Leu

(f ractions

is

also

observed work,

species

consists

cells

The effect

of leucine

In earlier

resolved

of tRNA Leu

(2,3,5,12).

contain

deprived

unique

and from

of tRNA Leu from

patterns

Nonstarved

tRNA from

graphically

synthesis

(4,5,7,12)

it

of leucine

strain

starvation is

isoacceptors

an additional 123-132).

was determined

chromato-

Frequently,

a minor,

starved

that

cells

the apparently

of subspecies

following

shown

while

major,

tRNA from

of a mixture

with

of E. coli

leucine

in the

by RPC-5 chromatography

treated

partial

which purifi-

(5).

unique

species

of leucine

315

tRNA is

also

observed

in rel-

cells

Vol. 73, No. 2,1976

BIOCHEMICAL

starved of arginine and in cells

AND BIOPHYSICAL

treated with chloramphenicol or puromycin (5).

The unique tRNAs produced under these different chromatographically formation.

similar,

RESEARCH COMMUNICATIONS

culturing

conditions are

suggesting a commonbiochemical basis for their

It was suggested (3-5, 12) that the unique species of tRNA from

leucine-starved

or chloramphenicol-treated

the normal isoacceptor of activity

cells were undermodified forms of

species, the appearance of which stemmedfrom the loss

of certain

of the base modifying enzymes. Direct sequence analyses Leu of the major unique tRNA and tRNAPhe from leucine-starved rel- cells (13 and Kitchingman and Fournier,

manuscripts in preparation)

tRNAphe from chloramphenicol-treated

cells

species are indeed, modification-deficient isoacceptors. MAphe

Relative

to the fully

species are deficient

(14) have revealed that the unique forms of the major, normally occurring

modified tRNAs, the unique tRNALeu and

in specific

dihydrouridine

Phe residues and the unique tRNA is additionally thiomethyl

are related,

in 4abu3U and the

adenosine (13).

While

that the unique and normal isoacceptors

it was not clear from earlier

forms are normal intermediates

and pseudouridine

deficient

moiety of 2-thiomethyl-N6-(A'-isopentenyl)

it has been established conclusively

and the unique

studies whether the hypomodified

in the tRNA biosynthetic

Suggestive evidence that a true precursor-product

process. relationship

exists for

the unique and normal species was derived from recovery studies in which the Leu relative amount of the unique species of tRNA and tRNAphe were observed to decrease upon restoration

of leucine to a culture

of leucine-starved

cells

(5)

or, in the case of tRNAPhe, removal of chloramphenicol from the growth medium (3) * The redistribution

of isoacceptor species occurred only slowly,

however,

a fact which could be explained by a slow rate of conversion or, rather, dilution

of the unique species by newly synthesized,

from a pulse-chase experiment indicated converted to a chromatographically

simple

fully

modified tRNA. Results Phe could be that the unique rel- tRNA

normal form (15). Leu To determine if the unique tRNA species could be converted to normal Leu and to gain more information about the post-transcriptional modification tRNAL

316

Vol. 73, No. 2, 1976

of tRNA,

BIOCHEMICAL

we have performed

tRNAs formed

during

distribution

of label

determined

at various

The results

a label-chase

leucine

times

show that

were

by a two step

with

the unique

32PO~3-,

of leucine

species

of tRNA

process

but,

Leu

species

to the starved

can,

that

rel-

and the

tRNALeU isoacceptor

restoration

the unique

in which

labeled

and normal

after

RESEARCH COMMUNICATIONS

experiment

starvation

in the unique

to tRNA Leu I

--in vivo

AND BIOPHYSICAL

in

fact,

culture.

be converted

the maturation

occurs

only

slowly. MATERIALS

AND METHODS:

benzoylated-DEAE

Materials

cellulose

obtained

to Method

from New England

Culture thr-,

(Whatman

according

and amino

Briefly,

twenty

--E. coli

experiment

millicuries

starvation.

After

supplemented At 0, 3.75

washed

and 5.25

extraction

the major

hours

Leu

10% polyacrylamide

gel

from

studies.

(16);

sources:

Schwarz-Mann; RPC-5 sorbent 32P (H3P0,+) was

(LP)

after for

were

cells

(17). were

his-,

leu-,

Conditions

previously

for

thi-,

culturing

( 4).

as described

previously

(15).

H PO were added to a 300 ml culture of 3 4 medium 20 to 30 minutes after the onset

6 hours

of starvation,

under

resuspension analysis Leu

.

Bulk

317

harvested fully

culturing

fresh

32P-tRNA

on benzoylated

in

were

medium,

conditions. one-third

Leu . tRNA

by electrophoresis

Approximately added

normal

on DEAE-cellulose

purified

cells

in 600 ml fresh,

in the

of the

tRNA

the

resuspended

and chromatography

by Dube et al.

leucine-starved

[32P]

and chromatography tRNA

from

CP79 (arg-,

was performed

of 32P-labeled

unique

described

in these

32P and incubated

was removed

Purification

mesh),

--et al.

E. coli

in LP medium,

medium minus

of the culture

phenol

of

phosophate

by centrifugation,

the following

from Reeve Angel;

have been described

CP79 in low

of leucine

DE-52),

C of Pearson

used

starvation

The label-chase

50-100

conditions.

was the strain acid

from

Nuclear.

and labeling

relA-)

obtained

(BD-cellulose,

diethylaminoethylcellulose was prepared

were

was prepared (4).

tRNALeU and I through

a slab

DEAE-cellulose

350 ug of unlabeled

the purification

by

process.

of as

bulk The

tRNA

BIOCHEMICAL

Vol. 73, No. 2, 1976

+Leu

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

375hrs

FRACTION

NUMBER

Leu Figure 2. Effect of restoring growth on the unique tRNA produced in relE. coli during leucieEustarvation. Labeling of the cells with [32P] PO;- and purification of tRNAU and tRNA$eu were performed as described in Methods. The tRNALeU was aminoacylated with [3H] leucine and fractionated by RPC-5 chromatography as described in Fig. 1. Radioactivity in each fraction was determined by counting portions of each fraction in 5 ml Liquifluor (New England Nuclear)/toluene/Triton X-100 (84:916:500) as described previously (5). The resulting RPC-5 elution profiles are shown. (0 - - - l ) r3H] leucyltRNA; (X X) [3zP] tRNA. tRNAp is identified as species 'I'; the unique tRNA sub-species are designated UI and UII. The material eluting in the region defined by fractions 120-130 corresponds to which is twiff co-purified to some extent with tRNAp and the unique tRNAs in the procedure used here. Analysis of the fingerprint of tRNA$ showed that this species is not related to tRNAILeu although there are a number of oligonucleotides common to both isoacceptors. Perhaps the label in tRNA@i is derived from the minor unique tRNALeu which elutes between tRNALeU and tRNAk5U (fractions 147-154) in the profile of total tRNALeU shown in srg. 1.

tRNA Leu from

BD-cellulose

by RPC-5 chromatography RPC-5 chromatography.

was aminoacylated as described

with

E3H] leucine

and fractionated

below.

tRNA samples

318

to be analyzed

by RPC-5 chromatography

Vol. 73, No. 2, 1976

were

BIOCHEMICAL

aminoacylated,

described

phenol

previously

equilibrated

extracted

(4,5).

in buffer

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and prepared

The sample

that

for

was applied

was 10 mM sodium

column to a 0.9

acetate,

and 0.4 NaCl and eluted with a linear 2 of 0.5M and 1.2M. Total volume of the gradient

were

collected.

The radioactivity

profiles

were

as

x 69 cm column

pH 4.5,

EDTA, 10 mM MgCl limits

analysis

1 mM disodium

NaCl gradient

with

was 600 ml;

determined

2 ml fractions

by scintillation

spectrometry. RESULTS AND DISCUSSION: shown

in

leucyl

Fig. [32P]

hours. the

2.

The results

The upper

panel

tRNALeU prepared

tRNA

is

of added

carrier

at least

three

87-92)

is

to the major eluted

tRNA from

unique

identified

species.

accounting

for

any,

label

was seen

only

a small

Incubation

of labeled

of the

32P-label

unique

species

amount increased

of label labeling

(22%)

cells species. coeluted

of tRNAp

for

indicates

six that

the occurrence

reflects eluting

species

(fractions

96-99)

minor

to that

eluting

of

(fractions

100-105)

A second,

and later

were

labeled total

during

of tRNAILeu

in fresh After with

the

32P activity (~10%)

under

the

medium 3.75

corresponds tRNA Leu

unique

of tRNA Leu and I

unique

species

starvation

are

normal

increased, and tRN$T

319

Little,

indicating

that

starvation

condition.

resulted

hours,

period,

respectively.

in major

a considerable

tRNAILeu , indicating

to a chromatographically in tRN Leu also 4rI

of leucine

13H]-

cells,

1.

of tRNAILeu was formed starved

of

respectively.

in the region

amount

profile

due to the presence

(fractions

and tRN
are

The 3H-profile,

of activity in Fig.

18% and 73% of the

of the

the pattern

peak

The earlier

tRN Leu and tRNA$y 4rI

starved

The earliest

intermediate

as tRNGy

Both

purity.

tRNA Leu shown

experiment

3H- and 32P-profiles

species.

The major

unique

cells

leucine-starved

isoacceptor

in a position

the major

high

chase

the RPC-5 elution

rel-

of the

of relatively

tRNAFU.

shows

from

The near-coincidence Leu

of the label

that

occurred

from at the

at best,

changes

in

proportion conversion

form had occurred.

doubling

if

The relative

18% to 36%. expense

of

The

of tRN Leu AU11

Vol. 73, No. 2,1976

BIOCHEMICAL

AND BIOPHYSICAL

which decreased from 72% to 41%of the total.

RESEARCH COMMUNICATIONS

The labeling

pattern

to change between 3.75 and 5.25 hours of recovery with tRNAr most highly labeled isoacceptor.

continued

becoming the

Over 42%of the 32P-label was associated wfth

Leu Leu tRNALeU I , while tRNAI,I and tRN%I1 accounted for 19%and 39%of the 32Pactivity respectively. The major unique species of tRNALeu can thus be converted to a form Leu chromatographically indistinguishable from tRNAI . However, as structural Leu analyses of the tRNAI produced in this experiment were not performed, it cannot be concluded that the 32P- labeled tRNALeU formed was completely modified. I The conversion of the unique species to tRNAy least two steps.

The labeling patterns

UI , then to tRNALeU The results I -

after

suggest that tRN$FF

also reveal that a significant

the unique species persisted even after time the number of viable cells

appears to have occurred in at

5.25 hours of recovery.

(estimated at better

six hours of starvation--ref.

was converted to proportion

of

During this

than 90%of the population

5) increased by 150%.

The slow rate of conversion could be interpreted to mean that the unique Leu species of tRNA are not proper substrates for the modifying enzymes which convert tRN$Fs to tRNAp in the biosynthesis reactions

involved

and that these species may not be normal intermediates

of tRNAy

.

Perhaps someof the nine base modification

Leu in the maturation of tRNAI occur in an ordered manner,

while others can occur at any time during the modification more of the ordered modifications

process.

If one or

occurs out of sequence, the resulting

may not be a good substrate for further

modification.

Modification

tRNA

of certain

bases may be accompanied by subtle changes in tRNA conformation which affect the formation of other modified nucleotides. be that certain of a larger, substrate.

of the base modification

Another,

related explanation would

reactions may usually occur at the level

precursor tRNA and, once cleaved, the mature-sized tRNA is a poor Yet another possibility

is that the hypomodified, unique species

mature slowly because they are not readily compartmentalization

available

of the modifying enzymes.

320

for modification

owing to

BIOCHEMICAL

Vol. 73, No. 2, 1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

A C C A

pG . C C - G G * C A - U A C G * c G * C U A

A

% U-D

GCG .

Gm

U

.

A

s Cd)

G .

G

A

D

T

.

CGC

G

U

c

U.A G

C * G U'A A-U G * Y C - G

G

A A C

. . . . . GGGGG

G

G

U G

ccccc

. G U.G c . C

Y-4

G A u

u

G* %'G

U

'AG

Figure 3. Structure of the major, unique leucine tRNA from relE. &. The structures of tRNApU and the major unique tRNALeU from leucize-starved cultures of --E. coli CP79 were determined according to the method of Brownlee (19 and will be described in detail elsewhere. and Sanger (18) and Griffin The structure shown is of tRNA k e" (17, 20). The arrows indicate the positions at which the unique reltRNALeh differs from fully modified tRNApu. (d) indicates a deficiency; the I in the Y loop is a mixture of II and I in both the reltRNA$eu and unique tRNALeU. G* is an unidentified derivative of G.

Results script

sequencing

in preparation)

differs Fig.

from

from

have

uridine

17 and pseudouridine

(II)

('i') 39.

to the anticodon

occurs

at position

38 in fully

position

for

residues

subfractions

of the

occurs

by the

species

unique

formation

that (UII

321

terminus

absence

of oligonucleotide the --in vivo

+ VI),

guanosine

G which

shift

then

(see

(D) 16 and

normally

G* 38 and 139 were

due to the

Results

tRNA indicate

5'

of dihydrouridines

of tRNAILeu , so the

is probably

of 016 first

the

(manu-

of tRNA Leu

species

of the modified tRNAILeu .

in the D-loop.

elsewhere

tRNALeU has an unmodified

instead modified

unique

38 and 39 from

in place

in our preparations

the unique

hydrouridine

occurs

in detail

the major

The unique

adjacent

deficient

that 16, 17,

residue

partially

to be presented

revealed

tR.NAILeu at positions

Unmodified

3).

studies

also

in elution of the two dianalyses

conversion

D17 (UI + 1).

of

probably

Vol. 73, No. 2, 1976

BIOCHEMICAL

These results provide direct relationship

RESEARCH COMMUNICATIONS

in vivo evidence for a precursor-product --

for the chromatographically

a normal isoacceptor. indicated

AND BIOPHYSICAL

As indicated

unique species of rel-

earlier,

a similar

tRNALeu and

relationship

has been

for the major unique and normal species of tRNAPhe from leucine

starved rel-

cells

(15).

ACKNOWLEDGEMENT: The study was supported in part by USPHSGrant GM19351 from the National Institute

of General Medical Sciences.

REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Littauer, U. Z., and Inouye, II., -Annu. Rev. Biochem., 62, 439-470 (1973). Waters, L. C., Biochem. Biophys. Res. Commun ., 3J, 296-304 (1969). Mann, M.B., and Huang, P. C., Biochemistry 12, 5289-5294 (1973). Fournier, M. J., and Peterkofsky, A., 2. Bacterial., 122, 538-548 (1975). Kitchingman, G. R., and Foumier, M. J., 2. Bacterial., 124, 1382-1394 (1975). Mann, M. B., and Huang, P. C., J. Bacterial., 118, 209-212 (1974). Chase, R., Tener, G. M., and Gillam, I. C., -Arch. Biochem. Biophys., 163, 306-317 (1974). Mandel, L. R., and Borek, E., Biochemistry, 2, 560-566 (1963). Powers, D. M., and Peterkofsky, A., Biochem. Biophys. Res. Commun.,46, 831-838 (1972). Harris, C. L., Titchener, E. B., and Cline, A. L., J. Bacterial., 100, 1322-1327 (1969). Nishimura, S., Taya, Y., Kuchino, Y., and Ohashi, Z., Biochem. Biophys. g. Commun.57, 702-708 (1974). Waters, L. C., Shugart, L., Yang, W., and Best, A. N., -Arch. ~ Biochem. Biophys., 156, 780-793 (1973). Kitchingman, G. R. and Fournier, M. J., -Fed. Proc. 35, 1615 (1976). Huang, P. C., and Mann, M. B., Biochemistry, 12, 4704-4710 (1974). Kitchingman, G. R., Webb, E., and Fournier, M. J., Biochemistry 12, 18481857 (1976). Pearson, R. L., Weiss, J. F. and Kelmers, A. D., Biochim. Biophys. u, 228, 770-774 (1971). Dube, S. K., Marcker, K. A., and Yudelevich, A., FEBSs., -,9 168-170 (1970). Brownlee, G. G., and Sanger, F., J. m. w., -,23 337-353 (1967). Griffin, B. E., FEBS&., -,15 165-168 (1971). Blank, H. U. andxl, D., Biochem. Biophys. Res. Commun.,-,43 1192-1197 (1972).

322