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Attenuation at nucleotide biosynthetic genes and amino acid biosynthetic operons of Escherichia coli
362
TIBS 11 - September 1986
Reviews Attenuation at nucleotide redu themchainohrateX This tightens the coupling between RNA biosynthetic genes and amino acid causes polymerase and the ribosomes and increased transcription of the biosynthetic operons of structural genes. The question to be analysed here is Escherichia how these two types of attenuators can respond in opposite ways to a close Kaj Frank densen, Fans Bonekamp and Peter Poulsen Transcription and translation are physically coupled in bacteria. The coupling ~ variable and regulates expression o f many genes. It influences premature termination o f m R N A chain synthesis - a process termed attenuation. In some cases attenuation is controlled primarily by the saturation o f R N A polymerase with nucleotides, at other genes the concentration ofaminoacyl-tRNAs is ofprimary importance.
As bacteria have no nucleus and no nuclear membrane to separate translation from transcription, the two processes are physically coupled (Fig. 1). This is exploited in bacteria for regulation of gene expression by a process termed attenuation, which is a mechanism for control of mRNA chain termination in front of a structural genek The basic idea in models accounting for this type of genetic control is that the 'naked' part of the growing transcript in front of the leading ribosome may adopt different configurations, hairpins or stem-loop structures, that influence the behaviour of R N A polymerase. Some may act as pause sites while others may terminate transcription I-5. Ribosomes which follow closely after R N A polymerase are believed to hinder sterically formation of such structures. Thereby the entire complex of RNA polymerase and ribosomes linked t°gether bY the m R N A chain determines whether or not transcription will be terminated3, 6. Elongation of protein chains and m R N A chains occurs at variable rates in Escherichia coli under the influence of gene structure and growth conditions7-tl . Relatively small changes of the global elongation rates may alter considerably the length of 'naked' R N A between R N A polymerase and the ribosomes (Fig. l) and thereby strongly influence secondary structure formation in the nascent m R N A chain. Regulation of gene expression by K. F. Jensen, F. Bonekamp and P. Poulsen are at the Institute of Biological Chemistry B, University of Copenhagen, SOlvgade 83, DK-1307 Copenhagen K, Denmark. (~1986, ElsevierSciencePublishersB.V.,Amsterdam
attenuation takes place at amino acid biosynthetic operons k12 and at certain genes involved in pyrimidine nucleotide biosynthesis 13-17. In the first case, the end product is a specific amino acid and the regulatory, modulating signal is the charging level of the cognate tRNA H. A low charging level of the tRNA is believed to reduce the rate of ribosome propagation at cognate codons and thereby to increase transcription past the attenuator. In the case of pyrimidine nucleotide biosynthesis, the end products of the pathway are UTP and CTP, which are used almost exclusively for R N A synthesis. A reduction in the cellular pool of UTP, which has a very high K m for binding at the elongation site on R N A polymeraselk is, is expected to
~ 1
-
coupling between R N A polymerase and the leading ribosome. We shall use the tryptophan operon as the only example of attenuation at amino acid biosynthetic operons, since these attenuators have been reviewed several times before1,12. The attenuators It is generally accepted that attenuation involves a Rho-independent transcription terminator positioned between the promoter and the structural gene(s) 3. Such a terminator is found both in the tryptophan leader (the 3 : 4 stem-loop, see later) and in front of the pyrBl and pyrE genes, which encode the subunits of aspartate transcarbamylase and orotate phosphoribosyltransferase, respectively. The three structures resemble each other closely and all consist of a GC-rich hairpin followed by a block of eight uridylate residues in the m R N A chain (Fig. 2). They terminate more than 95% of the transcriptsinvitrokX3,17. This termination
is influenced by translating ribosomes/n vivo, but the open reading frames for the leader translation end at very different positions relative to the three attenuators (Fig. 2).
It ¢ ~
]D
Fig. 1. Coupledtranscription and translation in bacteria; --, DNA ; ~, RNA; , protein.
0376 5067/86/$02.00
T l
TIBS 11 - September 1986
363 A
A
u C
C G
AUG. . . . . .
Start of trp
a9 . . . . . .
UGA. . . . . . . .
G c C c 43. . . . . . -G
dramatically lower expression of the two
O
G A fro attenuator
pyr genes (K. F. Jensen, unpublished).
c 6 G 6 CUUUUUUUU
End
leader peptide
u G o w e attenuator u A cA U G Gc cG CG UOA- - 7- - G CUUUUUUUU End GA
AUO. . . . . . . . . . . . . .
Start of
711. . . . . . . . . . . . . .
ortE
A
~am. . . . . . . . . . . . . . Start of oyrB leader oeptide
a6. . . . . . . . . . . . . .
u C pyrB attenuator G A G 6 G G eUUOOUUOU---lZ---UAA End
A c u C c e ¢ G
Fig. 2. Attenuators and leader translation for the trp, pyre and pyrBI operons of Escherichia coil The translated regions are shown in bold type. The open reading frame for leader translation ends 43 bp upstream of the trp attenuato#. The pyrE attenuator is located in the untranslated intercistronic space of a bicistronic operon (odE-pyrE). Translationofthe238-codonorfEgeneends7bpupstreamofthepyrEattenuator16, iv. ThepyrBI attenuator is situated inside a 44-codon open readingfrarne for a leader peptide t5-~7,21
Attenuation at pyrimidine nucleotide biosynthetic genes The levels of pyrBl and pyrE gene expression are inversely correlated to the cellular UTP poop 9. They also respond to changes in other nucleotide pools, but less dramatically than for UTp20. By use of gene fusions it was found that attenuation rather than promoter control is responsible for the vast majority of regulation by U T P (Ref. 21; P. Poulsen, unpublished). Insertion of small D N A fragments bearing the attenuator areas from the two genes (pyrB1 and pyrE) in the start of a lacZ gene, as shown for the pyrE gene in Fig. 3, gave rise to UTPregulated expression of the resulting hybrid pyr"lacZ gene transcribed from the/ac-promoter (Fig. 4). Modulation of attenuation, however, was dependent on the translation initiating at the original lacZ' start, now placed prior to the attenuators (Fig. 3). Significant transcription past the attenuator and regulation by the UTP pool was seen whenever the open reading flame extended close to or over the symmetric attenuator structure, and even if translation occurred in differe n t p h a s e s 22,23. However, if translation ended more than 32 nucleotide residues upstream of the symmetric attenuators, these functioned as permanent termination sites for transcription, regardless of the UTP p o o l 22,23.
Certain R N A polymerase mutations exhibiting a reduced rate of R N A chain elongation cause strongly enhanced levels ofpyrBl andpyrE gene expression, even when the cellular UTP pool is highIS,24, O n the other hand, mutations that reduce the rate of ribosome propagation, due to enhanced proof-reading 25,
The codon usage in the leader also influences transcription past the pyrE attenuator 9. By use of synthetic oligonucleotides a set of four plasmids was constructed, each containing one of the four combinations of C G T codons decoded by a m a j o r tRNA~rg species, and A G G codons decoded by a minor t R N A species 26. It was found that transcription past the pyrE attenuator increased by a factor of two each time a slowly translated A G G codon was replaced by a rapidly translated C G T codon. When the cellular UTP pool was kept low, however, the effect of codon composition in the leader vanished 9. Thus it seems that the length of m R N A between R N A polymerase and the first translating ribosome determines the frequency ofpyr gene transcription #z vivo. This is probably because a ribosome close after the transcribing R N A polymerase prevents the m R N A chain from adopting the terminating hairpin s t r u c t u r e at t h e a t t e n u a t o r .
Attenuation at amino acid biosynthetie operons Attenuation at amino acid biosynthetic operons is more complex than for the pyr genes since alternative and mutually exclusive conformations of the m R N A chain are involved I32. The tryptophan attenuator system is shown as an example in Fig. 5. It is striking that the reading frame of 14 codons for the leader peptide of this attenuator ends quite far (43 residues) upstream of the symmetric
!2" P?" ' [
A+~ orf E 1:"!::":~::]:::":;~:~:' I',11?:!i"!~)"!"~::)iii~i:::::~:!i::~iit!(;i :": ~ ~ " PL~ i c
' " "
]
J lac Z //
I
pp. LAC IIEP
pyr E
I
"~ ATN
-
]
1
//
]
Fig. 3. Cloning of the intercistronic orfE-pyrE region with the attenuator into a lacZ gene on a plasmid. A pyrE"laeZ hybrid gene is formed as well as a small artificial gene for a leader peptide. Transcription starts at the lac promoter and is subject to facultative termination at the pyrE attenuator as shown by the short and long mRNA transcripts beneath the construct 22. Note the unique cut sites for EcoR1 (E) and Pst/(P) in the leaderpeptide gene. These sites are used to vary the codon content during leader translation (see below). PLAC denotes the lac promoter; P t and 1>2are the promoters for pyrE operon transcription; A TN denotes the pyre
attenuatorwiththeopposingarrow-headsindicatingthesymmetricregions.A similarconstruetionwasmade for the attenuator of the pyrBI operon23.
364
T 1 B S 11 - S e p t e m b e r 1986
t (a)
/
/
30
•~
__
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form and to promote transcription of the structural genes of the operon. Thus, the tandem up codons ensure a response to the tryptophan supply, but they are not sufficient to secure specificity. The ribosomes may lag considerably behind RNA polymerase for other reasons, for instance if other amino acids are lacking. However, relief of attenuation in this system is known to be specific for tryptophan limitation, arginine starvation being the only exception I . To account for this we find another possible stemloop structure in the tryptophan leader, consisting of segments 1 and 2 basepaired with each other (Fig. 5a). The 1 : 2 structure is excluded by ribosomes stalled at, or downstream from, the trp codons. However, if the ribosome hesitares at a position upstream of the
./.
(b)
/
20 •
"~ z~ ~"
t / IPTG A ~aJ/f ^
t0
^
zx
Uracil
i_L i ~'~: ~./ OOe o ~
•
,k-"'• 0.2
04
•,.__J 0.6
- 02
i 04
i 0.6
No. of cells (OD4:~;) Fig.4.Expressi•nandregu•uti•n•fthepyrE'••acZhybridgene•nplasmidpKCLI•7.Thec•ntent•f]Lgalactosidase in the cMture is depicted as a function of the number of cells (0 D43a). Transcription of the hybrid gene was induced by addition of iso-propyl-J3-e-thiogalactoside(IPTG). The background strain requires exogenous pyrimidine for growth because of a mutation in pyrB. We have used either 5' UMP, uracil, or a combination thereof. When the cells grow with uracil the UTP pool is normal, but if they are fed UMP, the pool of UTP is low22. (a) A, uracil2Otzg/ml; ZX,5'UMPlOOizg/ml. (b)O, thecellsweregrowninthepresenceofS'UMPand at the indicated time uracil (20 t~g/rnl) was added; 0, the cells were grown in the presence of a small amount of uracil (0.5 izg/ml) and excess UMP (100 i.tg/ml).
terminator formed by basepairing between segments 3 and 4 (Refs 1, 27 and 28; Fig. 2). Ribosomes translating at a position so far upstream cannot be expected directly to interfere with the folding of this structure (see the preceding paragraph). Instead, ribosomes in the tryptophan leader 'communicate with' the attenuator, and hence with RNA polymerase, via another hairpin that can form by basepairing between segments 2 and 3 (Fig. 5b). This 2 : 3 hairpin is called the ant#terminator since it cannot co-exist with the transcription terminator due to competition for segment 3. The leader peptide gene ends just prior to the 2 : 3 hairpin, which may therefore be disrupted by ribosomes following closely after RNA polymerase. In that case, segment 3 is set free to basepair with segment 4 and form the terminator of transcription 1,12(Fig. 5). The principal difference between the function of the trp and the p y r attenuation systems is thus that a ribosome closely coupled to RNA polymerase in the up leader promotes transcript termination because it disrupts a structure which, if allowed to form, excludes the transcription terminator. For the pyrimidine systems such a ribosome relieves attenuation, because it interferes with the terminator itself. Another complexity of the trp attenuation system is that it responds specifically to the charging level of the tRNATw. In part, this task is accomplished by the occurrence of tandem tryptophan codons at a critical position in the leader
tandem trp codons the I : 2 stem-loop structure will form and destabilize the and-terminator because of competition for segment 21,29. The 1 : 2 hairpin structure thus protects the trp attenuator against modulation by false signals and provides specificity.
peptide (Fig. 5). If the ribosomes hefttare at this position, due to tryptophan limitation, while RNA polymerase continues, the ana-terminator is allowed to
Conclusions The fine tuning of coupling between transcription and translation can regu~
(a) Termination
3
~
UGA
/
(b)
Anti-termination
2
..,---r-..
~/////,..3, ~ ~
4 -7 OGA , ~ L
(c) Termination ~,/~f-),x 1 r-
_m
2
mr
Fig. 5. Effect of ribosome position on transcription past the trp-leader. The solid line indicates the symmetric attenuator segment. The numbers 1, 2, 3 and 4 refer to the segments in the mRNA chain involved in formation of regulatory hairpin structures (see texO. The tandem trp codons are indicated as short black bars. The circlesat the end of the mRNA chains symbolize RNA polymerase.
365
T I B S 11 - September 1986
late bacterial gene expression in very subtle ways depending on the primary gene structure. At amino acid biosynthetic operons these variables can be tuned to give attenuation responding specifically to the supply of a single
3 Platt, T. andBear, D.G.(1983) inGeneFunc-
amino acid. In case of pyrimidine nucleotide biosynthesis the coupling
5 Stroynowski,1., Kuroda, M. andYanofsky,C.
between transcription and translation may serve to balance the synthesis of UTP and CTP to the supply of amino acids. Thus the coupling responds not only to the cellular supply of nucleotides but also to the propagation rate of the ribosome. If the charging level of tRNAs in general is increased as a result of amino acids becoming available in the medium the cells will grow more rapidly and need more nucleotides for R N A synthesis30. Immediately this will cause the ribosomes to translate slightly faster, the coupling will be tighter, and the pyrimidine biosynthetic genes transcribed more frequently. Conversely, if amino acids are removed and the charging level of tRNAs falls, the ribosomes will lag behind R N A polymerase, and the attenuators will terminate a larger fraction of the m R N A chains prior to the pyr genes.
6 Petersen,S. (1984) EMBOJ. 3, 2895-2898 7 Adhya, S. and Gottesman, M. (1978) Annu. Rev. Biochem. 47,967-996 8 Petersen,S. (1985) in The Molecular Biology of BacterialGrowth (8chaecter,M., Neidhardt,F. c., Ingraham,J. L. and Kjelgaard,N. O., eds), pp. 13-20,Jones& Bartlett 9 Bonekamp,F., Andersen,H.D.,Christensen, T and Jensen, K. F. (1985) Nucleic Acids Res. 13, 4113-4123 10 Kassavetis, G. A. and Chamberlin, M.J. (1981) J. Biol. Chem. 256, 2777-2786 11 Kingston,R. E., Nierman, W. C. and Chamberlin, M. J. (1981) J. Biol. Chem. 2 5 6 , 2787-2797 12 Bauer, C. E., Carey, J., Kasper, L. M., Lynn, s.P., Waechter, D. A. and Gardner, J.F. (1983) in Gene Function in Prokaryotes (Beckwith,J., Davies, J. and Gallant, J.A., eds), pp. 65-89, Cold SpringHarbor Laboratory 13 Roof, W. D., Foltermann,K. F. and Wild, J. (1982) Mol. Gen. Genet. 187,391-400 14 Turnbough, C. L., Jr, Hicks, H. L. and Donahue, J. P. (1983) Proc. NatlAcad. Sci. USA 80, 368-372
References 1 Yanofsky, C.(1981)Nature289,751-758 2 Platt, T. (1981) Ce1124, 10-23
The
lysosome
tioninProkaryotes(Beekwith,J.,Davies, J. and
Gallant,J. A., eds), pp. 123-16l, ColdSpring HarborLaboratory 4 Farnham, P. J. and Platt, T. (1981) Nucleic Acids Res. 9,563-577 (1983)Proc.NatlAcad. Sci. USA80,2206-2210
membrane
The membrane around the lysosome is more than a cordon sanitaire, protecting the cytoplasm from the dangerous brew o f hydrolytic enzymes in these organdies. Its permeability pr•per•iesaretai••r-madet•enhancethee•ftdency•flys•s•mefuncti•n.Speciftctransp•rters exist in the membrane for certain metabolites,
John Lloyd and Susan Forsterare at the Department of Biological Sciences, University of Keele, Stafford. shireST55BG, UK.
NatlAcad.Sci. USA80,1207-1211
16 Poulsen, P., Jensen, K. F., Valentin-Hansen, P., Carlsson, P. and Lundberg, L. G. (1983) Eur. J. Biochem. 135,223-229 17 Poulsen,P., Bonekamp, F. and Jensen, K. F. (1984) EMBO J. 3, 1783-1790 18Jensen, K.F.,Fast, R.,Karlstrrm, O. andLarsen, J. N. (1986)J. Bacteriol. 166,857-865 19 Schwartz, M. and Neuhard, J. (1975) J. Bacteriol. 121,814-822 20 Jensen, K. F. (1979)J. Bacteriol. 138,731-738 21 Roland, K. L., Powell, F. E. and Turnbough, C.L., Jr (1985)J. Bacteriol. 163,991-999 22 Bonekamp,F., Clemmesen,K., Karlstrrm, O. andJensen, K.F.(1984)EMBOJ. 3, 2857-2861
23 Clemmesen,K.,Bonekamp,F., Karlstrrm,O. and Jensen, K. F. (1985) Mol. Gen. Genet. 201, 247-251 24 Jensen, K. F., Neuhard, J. and Schack, L. (1982) EMBO. J. I, 6%74 25 Ruusala, T., Andersson, D., Ehrenberg, M. and Kurland, C. G. (1984) EMBO, J. 3, 2575-2580 26 Grosjean, H. and Fiers, W. (1982) Gene 18, 19%209 27 Plan, T., Squires, C. and Yanofsky, C. (1976) J. MoL Biol. 103,411-420 28 DekeI-Gordetsky, L., Schoulaker-Schwartz, R. and Engelberg-Kulka, H. (1986) J. Bacteriol. 165, 1046-1048 29 Winkler, M. E., Mullis, K., Barnett, J., Stroynofsky,I. and Yanofsky, C. (1982) Proc. NatlAcad.Sci. USA79,2181-2185
30 MaalOe,O. andKjeldgaard, N.O.(1966)ControlofMacromolecularSynthesis, Benjamin
as a waste-dispossystem, there was
,John B. Lloyd and Susan Forster
In the early days of the lysosome, its membrane received as much attention as its enzymes. This was because the very existence of the organelle was revealed by clues deriving from the permeability properties ofitsmembrane. Theenzyme activities of intact lysosomes were 'latent', apparently because the membrane was impermeable to both enzymes and substrates;onlywhenthemembranewas ruptured by detergents or by freezethaw was full enzymic activity displayed, The impermeability of the lysosome membrane to the contained enzymes causes lysosomes to behave as simple
15 Navre, M. andSchachman, H.K.(1983)Proc.
osmometers: they are stable in an isoosmotic solution of a non-penetrating solute, but burst rapidly if suspended in a hypotonic solution, The lysosome membrane constitutes an important and effective barrier between the cytoplasm and the lysosomal contents. The need for this barrier is self-evident: the lysosomal enzymes could wreak havoc if they had unrestricted access to the cytoplasm. In the period when lysosomes were still thought of as 'suicide bags', pre-packaged destruction awaiting a programmed instruction to strike, their membranes could be considered simply as a cordon sa/'///a/'re. As it became clear that lysosomes have a more benign, if less exciting, role in the cell's economy, namely
increasing emphasis on the selective permeability of the membrane. While the membrane around the lysosome certainly keeps the enzymes from straying, the products of their digestive activity must be able to escape into the cytosol. Otherwise the contents of the lysosomes would constantly increase, and the metabolic pathways of the cytosol would be denied the products of lysosomal catabolism. The constant flow of metabolites out of the lysosome is of course fueled by the constant influx of macromolecules, which arrive by mechanisms that avoid transmembrane passage I. Pinocytosis and phagocytosis introduce material arising outside the cell, while autophagy, crinophagy, and the still controversial microautophagy bringin organelles and macromolecules from the cytosol. But translocation of metabolites from cytosol into the lysosome is important in some special cases. Heparan sulphate is apparently resistant to intralysosomal degradation until its glucosamine moieties are acetylated in the lysosome by cytoplasmderived acetyl coenzyme A (see below). For some years there has been a
~ 1986,ElsevierSciencePublishersB.V.,Amsterdam 0376-5067/861502.00
TIBS 11 - September 1986
Reviews Attenuation at nucleotide redu themchainohrateX This tightens the coupling between RNA biosynthetic genes and amino acid causes polymerase and the ribosomes and increased transcription of the biosynthetic operons of structural genes. The question to be analysed here is Escherichia how these two types of attenuators can respond in opposite ways to a close Kaj Frank densen, Fans Bonekamp and Peter Poulsen Transcription and translation are physically coupled in bacteria. The coupling ~ variable and regulates expression o f many genes. It influences premature termination o f m R N A chain synthesis - a process termed attenuation. In some cases attenuation is controlled primarily by the saturation o f R N A polymerase with nucleotides, at other genes the concentration ofaminoacyl-tRNAs is ofprimary importance.
As bacteria have no nucleus and no nuclear membrane to separate translation from transcription, the two processes are physically coupled (Fig. 1). This is exploited in bacteria for regulation of gene expression by a process termed attenuation, which is a mechanism for control of mRNA chain termination in front of a structural genek The basic idea in models accounting for this type of genetic control is that the 'naked' part of the growing transcript in front of the leading ribosome may adopt different configurations, hairpins or stem-loop structures, that influence the behaviour of R N A polymerase. Some may act as pause sites while others may terminate transcription I-5. Ribosomes which follow closely after R N A polymerase are believed to hinder sterically formation of such structures. Thereby the entire complex of RNA polymerase and ribosomes linked t°gether bY the m R N A chain determines whether or not transcription will be terminated3, 6. Elongation of protein chains and m R N A chains occurs at variable rates in Escherichia coli under the influence of gene structure and growth conditions7-tl . Relatively small changes of the global elongation rates may alter considerably the length of 'naked' R N A between R N A polymerase and the ribosomes (Fig. l) and thereby strongly influence secondary structure formation in the nascent m R N A chain. Regulation of gene expression by K. F. Jensen, F. Bonekamp and P. Poulsen are at the Institute of Biological Chemistry B, University of Copenhagen, SOlvgade 83, DK-1307 Copenhagen K, Denmark. (~1986, ElsevierSciencePublishersB.V.,Amsterdam
attenuation takes place at amino acid biosynthetic operons k12 and at certain genes involved in pyrimidine nucleotide biosynthesis 13-17. In the first case, the end product is a specific amino acid and the regulatory, modulating signal is the charging level of the cognate tRNA H. A low charging level of the tRNA is believed to reduce the rate of ribosome propagation at cognate codons and thereby to increase transcription past the attenuator. In the case of pyrimidine nucleotide biosynthesis, the end products of the pathway are UTP and CTP, which are used almost exclusively for R N A synthesis. A reduction in the cellular pool of UTP, which has a very high K m for binding at the elongation site on R N A polymeraselk is, is expected to
~ 1
-
coupling between R N A polymerase and the leading ribosome. We shall use the tryptophan operon as the only example of attenuation at amino acid biosynthetic operons, since these attenuators have been reviewed several times before1,12. The attenuators It is generally accepted that attenuation involves a Rho-independent transcription terminator positioned between the promoter and the structural gene(s) 3. Such a terminator is found both in the tryptophan leader (the 3 : 4 stem-loop, see later) and in front of the pyrBl and pyrE genes, which encode the subunits of aspartate transcarbamylase and orotate phosphoribosyltransferase, respectively. The three structures resemble each other closely and all consist of a GC-rich hairpin followed by a block of eight uridylate residues in the m R N A chain (Fig. 2). They terminate more than 95% of the transcriptsinvitrokX3,17. This termination
is influenced by translating ribosomes/n vivo, but the open reading frames for the leader translation end at very different positions relative to the three attenuators (Fig. 2).
It ¢ ~
]D
Fig. 1. Coupledtranscription and translation in bacteria; --, DNA ; ~, RNA; , protein.
0376 5067/86/$02.00
T l
TIBS 11 - September 1986
363 A
A
u C
C G
AUG. . . . . .
Start of trp
a9 . . . . . .
UGA. . . . . . . .
G c C c 43. . . . . . -G
dramatically lower expression of the two
O
G A fro attenuator
pyr genes (K. F. Jensen, unpublished).
c 6 G 6 CUUUUUUUU
End
leader peptide
u G o w e attenuator u A cA U G Gc cG CG UOA- - 7- - G CUUUUUUUU End GA
AUO. . . . . . . . . . . . . .
Start of
711. . . . . . . . . . . . . .
ortE
A
~am. . . . . . . . . . . . . . Start of oyrB leader oeptide
a6. . . . . . . . . . . . . .
u C pyrB attenuator G A G 6 G G eUUOOUUOU---lZ---UAA End
A c u C c e ¢ G
Fig. 2. Attenuators and leader translation for the trp, pyre and pyrBI operons of Escherichia coil The translated regions are shown in bold type. The open reading frame for leader translation ends 43 bp upstream of the trp attenuato#. The pyrE attenuator is located in the untranslated intercistronic space of a bicistronic operon (odE-pyrE). Translationofthe238-codonorfEgeneends7bpupstreamofthepyrEattenuator16, iv. ThepyrBI attenuator is situated inside a 44-codon open readingfrarne for a leader peptide t5-~7,21
Attenuation at pyrimidine nucleotide biosynthetic genes The levels of pyrBl and pyrE gene expression are inversely correlated to the cellular UTP poop 9. They also respond to changes in other nucleotide pools, but less dramatically than for UTp20. By use of gene fusions it was found that attenuation rather than promoter control is responsible for the vast majority of regulation by U T P (Ref. 21; P. Poulsen, unpublished). Insertion of small D N A fragments bearing the attenuator areas from the two genes (pyrB1 and pyrE) in the start of a lacZ gene, as shown for the pyrE gene in Fig. 3, gave rise to UTPregulated expression of the resulting hybrid pyr"lacZ gene transcribed from the/ac-promoter (Fig. 4). Modulation of attenuation, however, was dependent on the translation initiating at the original lacZ' start, now placed prior to the attenuators (Fig. 3). Significant transcription past the attenuator and regulation by the UTP pool was seen whenever the open reading flame extended close to or over the symmetric attenuator structure, and even if translation occurred in differe n t p h a s e s 22,23. However, if translation ended more than 32 nucleotide residues upstream of the symmetric attenuators, these functioned as permanent termination sites for transcription, regardless of the UTP p o o l 22,23.
Certain R N A polymerase mutations exhibiting a reduced rate of R N A chain elongation cause strongly enhanced levels ofpyrBl andpyrE gene expression, even when the cellular UTP pool is highIS,24, O n the other hand, mutations that reduce the rate of ribosome propagation, due to enhanced proof-reading 25,
The codon usage in the leader also influences transcription past the pyrE attenuator 9. By use of synthetic oligonucleotides a set of four plasmids was constructed, each containing one of the four combinations of C G T codons decoded by a m a j o r tRNA~rg species, and A G G codons decoded by a minor t R N A species 26. It was found that transcription past the pyrE attenuator increased by a factor of two each time a slowly translated A G G codon was replaced by a rapidly translated C G T codon. When the cellular UTP pool was kept low, however, the effect of codon composition in the leader vanished 9. Thus it seems that the length of m R N A between R N A polymerase and the first translating ribosome determines the frequency ofpyr gene transcription #z vivo. This is probably because a ribosome close after the transcribing R N A polymerase prevents the m R N A chain from adopting the terminating hairpin s t r u c t u r e at t h e a t t e n u a t o r .
Attenuation at amino acid biosynthetie operons Attenuation at amino acid biosynthetic operons is more complex than for the pyr genes since alternative and mutually exclusive conformations of the m R N A chain are involved I32. The tryptophan attenuator system is shown as an example in Fig. 5. It is striking that the reading frame of 14 codons for the leader peptide of this attenuator ends quite far (43 residues) upstream of the symmetric
!2" P?" ' [
A+~ orf E 1:"!::":~::]:::":;~:~:' I',11?:!i"!~)"!"~::)iii~i:::::~:!i::~iit!(;i :": ~ ~ " PL~ i c
' " "
]
J lac Z //
I
pp. LAC IIEP
pyr E
I
"~ ATN
-
]
1
//
]
Fig. 3. Cloning of the intercistronic orfE-pyrE region with the attenuator into a lacZ gene on a plasmid. A pyrE"laeZ hybrid gene is formed as well as a small artificial gene for a leader peptide. Transcription starts at the lac promoter and is subject to facultative termination at the pyrE attenuator as shown by the short and long mRNA transcripts beneath the construct 22. Note the unique cut sites for EcoR1 (E) and Pst/(P) in the leaderpeptide gene. These sites are used to vary the codon content during leader translation (see below). PLAC denotes the lac promoter; P t and 1>2are the promoters for pyrE operon transcription; A TN denotes the pyre
attenuatorwiththeopposingarrow-headsindicatingthesymmetricregions.A similarconstruetionwasmade for the attenuator of the pyrBI operon23.
364
T 1 B S 11 - S e p t e m b e r 1986
t (a)
/
/
30
•~
__
/ ~_
form and to promote transcription of the structural genes of the operon. Thus, the tandem up codons ensure a response to the tryptophan supply, but they are not sufficient to secure specificity. The ribosomes may lag considerably behind RNA polymerase for other reasons, for instance if other amino acids are lacking. However, relief of attenuation in this system is known to be specific for tryptophan limitation, arginine starvation being the only exception I . To account for this we find another possible stemloop structure in the tryptophan leader, consisting of segments 1 and 2 basepaired with each other (Fig. 5a). The 1 : 2 structure is excluded by ribosomes stalled at, or downstream from, the trp codons. However, if the ribosome hesitares at a position upstream of the
./.
(b)
/
20 •
"~ z~ ~"
t / IPTG A ~aJ/f ^
t0
^
zx
Uracil
i_L i ~'~: ~./ OOe o ~
•
,k-"'• 0.2
04
•,.__J 0.6
- 02
i 04
i 0.6
No. of cells (OD4:~;) Fig.4.Expressi•nandregu•uti•n•fthepyrE'••acZhybridgene•nplasmidpKCLI•7.Thec•ntent•f]Lgalactosidase in the cMture is depicted as a function of the number of cells (0 D43a). Transcription of the hybrid gene was induced by addition of iso-propyl-J3-e-thiogalactoside(IPTG). The background strain requires exogenous pyrimidine for growth because of a mutation in pyrB. We have used either 5' UMP, uracil, or a combination thereof. When the cells grow with uracil the UTP pool is normal, but if they are fed UMP, the pool of UTP is low22. (a) A, uracil2Otzg/ml; ZX,5'UMPlOOizg/ml. (b)O, thecellsweregrowninthepresenceofS'UMPand at the indicated time uracil (20 t~g/rnl) was added; 0, the cells were grown in the presence of a small amount of uracil (0.5 izg/ml) and excess UMP (100 i.tg/ml).
terminator formed by basepairing between segments 3 and 4 (Refs 1, 27 and 28; Fig. 2). Ribosomes translating at a position so far upstream cannot be expected directly to interfere with the folding of this structure (see the preceding paragraph). Instead, ribosomes in the tryptophan leader 'communicate with' the attenuator, and hence with RNA polymerase, via another hairpin that can form by basepairing between segments 2 and 3 (Fig. 5b). This 2 : 3 hairpin is called the ant#terminator since it cannot co-exist with the transcription terminator due to competition for segment 3. The leader peptide gene ends just prior to the 2 : 3 hairpin, which may therefore be disrupted by ribosomes following closely after RNA polymerase. In that case, segment 3 is set free to basepair with segment 4 and form the terminator of transcription 1,12(Fig. 5). The principal difference between the function of the trp and the p y r attenuation systems is thus that a ribosome closely coupled to RNA polymerase in the up leader promotes transcript termination because it disrupts a structure which, if allowed to form, excludes the transcription terminator. For the pyrimidine systems such a ribosome relieves attenuation, because it interferes with the terminator itself. Another complexity of the trp attenuation system is that it responds specifically to the charging level of the tRNATw. In part, this task is accomplished by the occurrence of tandem tryptophan codons at a critical position in the leader
tandem trp codons the I : 2 stem-loop structure will form and destabilize the and-terminator because of competition for segment 21,29. The 1 : 2 hairpin structure thus protects the trp attenuator against modulation by false signals and provides specificity.
peptide (Fig. 5). If the ribosomes hefttare at this position, due to tryptophan limitation, while RNA polymerase continues, the ana-terminator is allowed to
Conclusions The fine tuning of coupling between transcription and translation can regu~
(a) Termination
3
~
UGA
/
(b)
Anti-termination
2
..,---r-..
~/////,..3, ~ ~
4 -7 OGA , ~ L
(c) Termination ~,/~f-),x 1 r-
_m
2
mr
Fig. 5. Effect of ribosome position on transcription past the trp-leader. The solid line indicates the symmetric attenuator segment. The numbers 1, 2, 3 and 4 refer to the segments in the mRNA chain involved in formation of regulatory hairpin structures (see texO. The tandem trp codons are indicated as short black bars. The circlesat the end of the mRNA chains symbolize RNA polymerase.
365
T I B S 11 - September 1986
late bacterial gene expression in very subtle ways depending on the primary gene structure. At amino acid biosynthetic operons these variables can be tuned to give attenuation responding specifically to the supply of a single
3 Platt, T. andBear, D.G.(1983) inGeneFunc-
amino acid. In case of pyrimidine nucleotide biosynthesis the coupling
5 Stroynowski,1., Kuroda, M. andYanofsky,C.
between transcription and translation may serve to balance the synthesis of UTP and CTP to the supply of amino acids. Thus the coupling responds not only to the cellular supply of nucleotides but also to the propagation rate of the ribosome. If the charging level of tRNAs in general is increased as a result of amino acids becoming available in the medium the cells will grow more rapidly and need more nucleotides for R N A synthesis30. Immediately this will cause the ribosomes to translate slightly faster, the coupling will be tighter, and the pyrimidine biosynthetic genes transcribed more frequently. Conversely, if amino acids are removed and the charging level of tRNAs falls, the ribosomes will lag behind R N A polymerase, and the attenuators will terminate a larger fraction of the m R N A chains prior to the pyr genes.
6 Petersen,S. (1984) EMBOJ. 3, 2895-2898 7 Adhya, S. and Gottesman, M. (1978) Annu. Rev. Biochem. 47,967-996 8 Petersen,S. (1985) in The Molecular Biology of BacterialGrowth (8chaecter,M., Neidhardt,F. c., Ingraham,J. L. and Kjelgaard,N. O., eds), pp. 13-20,Jones& Bartlett 9 Bonekamp,F., Andersen,H.D.,Christensen, T and Jensen, K. F. (1985) Nucleic Acids Res. 13, 4113-4123 10 Kassavetis, G. A. and Chamberlin, M.J. (1981) J. Biol. Chem. 256, 2777-2786 11 Kingston,R. E., Nierman, W. C. and Chamberlin, M. J. (1981) J. Biol. Chem. 2 5 6 , 2787-2797 12 Bauer, C. E., Carey, J., Kasper, L. M., Lynn, s.P., Waechter, D. A. and Gardner, J.F. (1983) in Gene Function in Prokaryotes (Beckwith,J., Davies, J. and Gallant, J.A., eds), pp. 65-89, Cold SpringHarbor Laboratory 13 Roof, W. D., Foltermann,K. F. and Wild, J. (1982) Mol. Gen. Genet. 187,391-400 14 Turnbough, C. L., Jr, Hicks, H. L. and Donahue, J. P. (1983) Proc. NatlAcad. Sci. USA 80, 368-372
References 1 Yanofsky, C.(1981)Nature289,751-758 2 Platt, T. (1981) Ce1124, 10-23
The
lysosome
tioninProkaryotes(Beekwith,J.,Davies, J. and
Gallant,J. A., eds), pp. 123-16l, ColdSpring HarborLaboratory 4 Farnham, P. J. and Platt, T. (1981) Nucleic Acids Res. 9,563-577 (1983)Proc.NatlAcad. Sci. USA80,2206-2210
membrane
The membrane around the lysosome is more than a cordon sanitaire, protecting the cytoplasm from the dangerous brew o f hydrolytic enzymes in these organdies. Its permeability pr•per•iesaretai••r-madet•enhancethee•ftdency•flys•s•mefuncti•n.Speciftctransp•rters exist in the membrane for certain metabolites,
John Lloyd and Susan Forsterare at the Department of Biological Sciences, University of Keele, Stafford. shireST55BG, UK.
NatlAcad.Sci. USA80,1207-1211
16 Poulsen, P., Jensen, K. F., Valentin-Hansen, P., Carlsson, P. and Lundberg, L. G. (1983) Eur. J. Biochem. 135,223-229 17 Poulsen,P., Bonekamp, F. and Jensen, K. F. (1984) EMBO J. 3, 1783-1790 18Jensen, K.F.,Fast, R.,Karlstrrm, O. andLarsen, J. N. (1986)J. Bacteriol. 166,857-865 19 Schwartz, M. and Neuhard, J. (1975) J. Bacteriol. 121,814-822 20 Jensen, K. F. (1979)J. Bacteriol. 138,731-738 21 Roland, K. L., Powell, F. E. and Turnbough, C.L., Jr (1985)J. Bacteriol. 163,991-999 22 Bonekamp,F., Clemmesen,K., Karlstrrm, O. andJensen, K.F.(1984)EMBOJ. 3, 2857-2861
23 Clemmesen,K.,Bonekamp,F., Karlstrrm,O. and Jensen, K. F. (1985) Mol. Gen. Genet. 201, 247-251 24 Jensen, K. F., Neuhard, J. and Schack, L. (1982) EMBO. J. I, 6%74 25 Ruusala, T., Andersson, D., Ehrenberg, M. and Kurland, C. G. (1984) EMBO, J. 3, 2575-2580 26 Grosjean, H. and Fiers, W. (1982) Gene 18, 19%209 27 Plan, T., Squires, C. and Yanofsky, C. (1976) J. MoL Biol. 103,411-420 28 DekeI-Gordetsky, L., Schoulaker-Schwartz, R. and Engelberg-Kulka, H. (1986) J. Bacteriol. 165, 1046-1048 29 Winkler, M. E., Mullis, K., Barnett, J., Stroynofsky,I. and Yanofsky, C. (1982) Proc. NatlAcad.Sci. USA79,2181-2185
30 MaalOe,O. andKjeldgaard, N.O.(1966)ControlofMacromolecularSynthesis, Benjamin
as a waste-dispossystem, there was
,John B. Lloyd and Susan Forster
In the early days of the lysosome, its membrane received as much attention as its enzymes. This was because the very existence of the organelle was revealed by clues deriving from the permeability properties ofitsmembrane. Theenzyme activities of intact lysosomes were 'latent', apparently because the membrane was impermeable to both enzymes and substrates;onlywhenthemembranewas ruptured by detergents or by freezethaw was full enzymic activity displayed, The impermeability of the lysosome membrane to the contained enzymes causes lysosomes to behave as simple
15 Navre, M. andSchachman, H.K.(1983)Proc.
osmometers: they are stable in an isoosmotic solution of a non-penetrating solute, but burst rapidly if suspended in a hypotonic solution, The lysosome membrane constitutes an important and effective barrier between the cytoplasm and the lysosomal contents. The need for this barrier is self-evident: the lysosomal enzymes could wreak havoc if they had unrestricted access to the cytoplasm. In the period when lysosomes were still thought of as 'suicide bags', pre-packaged destruction awaiting a programmed instruction to strike, their membranes could be considered simply as a cordon sa/'///a/'re. As it became clear that lysosomes have a more benign, if less exciting, role in the cell's economy, namely
increasing emphasis on the selective permeability of the membrane. While the membrane around the lysosome certainly keeps the enzymes from straying, the products of their digestive activity must be able to escape into the cytosol. Otherwise the contents of the lysosomes would constantly increase, and the metabolic pathways of the cytosol would be denied the products of lysosomal catabolism. The constant flow of metabolites out of the lysosome is of course fueled by the constant influx of macromolecules, which arrive by mechanisms that avoid transmembrane passage I. Pinocytosis and phagocytosis introduce material arising outside the cell, while autophagy, crinophagy, and the still controversial microautophagy bringin organelles and macromolecules from the cytosol. But translocation of metabolites from cytosol into the lysosome is important in some special cases. Heparan sulphate is apparently resistant to intralysosomal degradation until its glucosamine moieties are acetylated in the lysosome by cytoplasmderived acetyl coenzyme A (see below). For some years there has been a
~ 1986,ElsevierSciencePublishersB.V.,Amsterdam 0376-5067/861502.00