Ribosomal RNA and peptidyl-tRNA hydrolase: a peptide chain termination model for lambda bar RNA inhibition

Biochimie (1991) 73, 1573-1578

1573

© Socirt6 frangaise de biochimie et biologic molrculaire / Elsevier, Paris

Ribosomal RNA and peptidyl-tRNA hydrolase: a peptide chain termination model for iambda bar RNA inhibition E J M u r g o l a l, G G u a m e r o s 2 t Department of Molecular Genetics, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030 USA; 2Departmento de Genetica y Biologia Molecular, Centro de lnvestigacion y de Estudios Avanzados del IPN, Apostado Postal 14-740, Mexico DF 07000

(Received l November 1991; accepted 6 November 1991)

Summary - - We propose here a model to explain the inhibition of bacteriophage lambda (~.) vegetative growth and the killing of E coli cells defective in peptidyl-tRNA hydrolase (Pth) by ~, bar RNA. The model suggests that bar RNA, which contains a characteristic UGA triplet, base-pairs in an anti-parallel fashion with the t 199-1205 region of E coli 16S rRNA. In doing so, it prevents the

required functioning of that region of 16S rRNA in UGA-specific peptide chain termination. Pth is implicated in peptide chain termination because a defect in Pth is required for the achievement of the bar R N A inhibitory effects. We make certain predictions that flow from the model, predictions involving suppression of nonsense mutations, and present preliminary experimental results that demonstrate the fulfillment of those predictions. codon recognition / 16S rRNA / nonsense suppression / bar/16S synergism / antisense RNA target

Introduction

It was previously observed that, in peptidyl-tRNA hydrolase (Pth)-defective cells, transcription o f the b a r region o f bacteriophage lambda (~.) led to inhibition o f ~, vegetative growth. Furthermore, when b a r + was cloned into a high copy plasmid, the presence of the b a r + ~lasmid was lethal for Pth- (but not Pth +) cells. To explain these and related observations, we propose here a model that points to a ribosomal R N A nucleotide sequence ~ a target for anti-parallel base pairing with ~, b a r RNA. The model also implicates peptidyl-tRNA hydrolase in the normal peptide chain termination process. We state a few basic predictions of the model and present preliminary evidence for the fulfilment o f those predictions. Finally, we discuss some ramifications o f the model.

not [4]. The plasmid phenomenon closely parallels the phage behavior (table I)0 but during phage infection, cell lethality is not required for inhibition o f ~, growth (D Vazquez and G Guameros, unpublished results). It- _ L 1 rr~ ..... . r ~ - - ¢ ' : ~ - - lull ~ 4 ~ .W . . .I'I| I, ¢d ,-i- L.~. ./.l.a I K ; O LU - -tu ]l .IJ~I.NT/ ' ~Ak i s necessaa-y u u u l lll~Llli~gu,~lllJtlVO'll to prevent growth o f ~, and for b a r ~ plasmid lethality to E coli [3, 4]. The magnitude o f transcription through barI and b a r l I correlates with the degree o f Rap inhibition. After transcription induction, Bar* RNA accumulates much more rapidly than Bar- RNA and exhibits greater stability (3.5 min half-life versus less than 1 min) in Rap- bacteria [6]. Table I. Summary of k Bar effects (adapted from [5]). E coli strain

~, vegetative growth at 39°C bar +

T h e p h e n o m e n o n o f l a m b d a bar inhibition T h e rap mutant o f E coli is unable to support the

growth o f bacteriophage ~ [1, 2]. Phage mutations that o v e r c o m e the rap effect have been mapped to different sites in the phage genome, and two o f those sites were designated barI and barII [3]. Plasmids containing wild-type ~, b a r D N A segments kill rap mutant cells, but plasmids with mutant b a r regions do

A bar-

Growth o f transformed cellsa at 40°C p bar + p bar-

pth +

+b

+

+

+

pth-C

_b

+

_

+

abar+ and bar- regions of k were cloned under the transcriptional control of E coli pGal promoter, b+, represents

proper formation of plaques: -, absence of plaques. CThe pth- was the thermosensitive mutant pth(ts). This strain

grows at 40°C, although poorly.

1574

EJ Murgola,G Gaarneros

The implication of translation in these events came from two findings. First, the b a r ~ lethai':t,~' t,~ rap mutants was accompanied by inhibition of protein synthesis but not of RNA synthes~s [6]. Second was the demonstration that the rap gene is pth, the gene that encodes peptidyl-tRNA hydroiase [5]. Pth cleaves peptidyl-tRNA to yield free peptides and tRNA, and it has been proposed that the natural substrates for the hydrolase are peptidyl-tRNAs that drop off the ribosomes during protein synthesis [7, 8]. When bacteria carrying the thermosensitive Pth mutation pth(ts) are shifted to 43°C, one result is cessation of protein synthesis and another is accumulation of peptidyltRNAs, which are considered to somehow block protein synthesis [8]. The possible role of this enzyme in the ribosome-bound hydrolysis step of peptide cain termination was seriously considered, but extensive work failed to turn up any evidence to link Pth with peptide chain termination [8-10]. A comparison of the ~ barI and barlI regions revealed a nearly identical sequence (14 of 16 bp), which include an inverted repeat. The following is the barI RNA sequence with the homologous (to barlI) nucleotides italicized and ,~,e inverted repeat underscored. -: (bard 5 ' - A A U G A U A U A A A U A U C A A U A U A - 3 ' The 14 identical positions may be required, at least indirectly, for Bar RNA activity since the three different Bar- mutations that have been sequenced represent nucleotide transitions at one of those positions. Furthermore, for barI, a funcLonal sequence for killing Rap- cells has been detemuned to be the 21-bp sequence shown above [4]. In this paper, we present a unifying model that explains the observations to date~ a model that implicates Bar RbrA and Pth in peptide chain termination and suggests 16S rRNA as the target for Bar RNA activity.

A termination model and some basic predictions The model comprises two major parts. In the first pa~-t, we propose that Bar RNA can interfere with UGA termination by achieving anti-parallel base-pairing with the 1199 to 1205 sequence (5'-UCAUCAU-3') of 16S-RNA. In the second part, we suggest that Pth is involved ha peptide chain termination and that mutant Pth causes a defect in the termination process. We imagine that the effect of the mutant Pth in the presence of Bar RNA is either to allow the Bar-16S base-pairing to occur or to somehow exacerbate the effects of the RNA-RNA interaction. The suggestion of the Bar RNA:16S rRNA interaction was prompted first by the presence of 5'-UGA3' triplets in bar sequences and the involvement of nucleotides 1199 to 1204 in the helix 34 region of 16S

rRNA in UGA specific termination (11-14). In all three bar mutants sequenced so far, the mutations occurred within the 14 nucleotides that are the same in barI and barlI, one of them, in fact, having occurred in the middle of the UGA triplet composed of nucleotides 3 to 5 in the barI sequence given above! The possibility also exists that a 4 base pair interaction occurs between the sequence 5'-AUGA-3' in Bar RNA and nucleotides 1199 to 1202 and/or 1202 to 1205 in 16S rRNA, especially in the case of ~. mRNA translation. It is worth noting that the lambda genome has 15 genes that end in AUGA. For 13 of those, it is known that the AUG is the initiation signal for an overlapping gene [15]. Alternatively, a 5 base-pair interaction could occur between the 5'-AUGAU-3' sequence of Bar RNA and the 16S rRNA nucleotides 1201 to 1205 (5'-AUCAU-3') and possibly also 1198 to 1202 (5'-GUCAU-3'), if one allows for a G-U pair. The thought of Pth involvement in termination was less obvious and less direct. It was more than simply a 'leap of faith', but it was clearly based on guilt by association! Although there has been no direct evidence implicating Pth in ribosome-dependent chain termination, there has also been no unequivocal evidence precluding it from that function. The current model still allows for Pth to perform the scavenger function documented by Menninger and his coworkers (see, for example [16] and references therein), but it suggests a further role, what could indeed be the essential role, in peptide chain termination. In any case, the fundamental premises of the model, implicating Pth in termination and Bar RNA in the same process by way of base-pairing with rRNA, are open to a few basic, testable predictions and also t,~ more ;n,,,h,,A h.,, . . . I.. / I I ~ -,:-,:--. . . . . . ~,~me .. A...uzv~,u, U U L ,oo,~h, LV~.*i~l,O.t.Tt~, UII.~LIUIIS. The following are presented as two basic predictions of the model: 1)~ a), In Pth + cells (ie wild-type E coli), the Bar + plasmid should exhibit codon-specific nonsense suppression, that is, it should suppress UGA- but not UAA- or UAG-mutations; b), Bar- plasmids should not suppress UGA; c), bar + should suppress synergistically with some helix 34 mutants involved in termination; 2), at some temperat'lre(s), pth(ts), a temperature-sensitive Pth mutant [8], should suppress nonsense mutations of all three types (UGA, UAA, and UAG). Preliminary results of experiments addressing these predictions are summarized in the next section.

Experimental support for the basic predictions The first prediction The basis for the first prediction is the major proposal of the model, namely that bar RNA interferes with normal peptide chain termination by antiparallel base-

Model for lambda Bar RNA inhibition

plasmid'). To test for nonsense suppression we used different trpA mutant strains, each containing a trpA nonsense mutation caused by one of the three termination codons (UGA, UAA, UAG) present at one of four positions in the trpA mRNA, at codon 15, 211, 234, or 243. The strains were transtbrmed with each o f the 3 plasmids. The ampicillin resistant (Ampg) transformants were single-colony isolated and then tested for suppression (Trp + vs Trp-). At two o f the four positions, 211 and 234, no suppression was observed with any o f the three termination codons. Suppression o f U G A by bat -~ was seen, however, at positions 15 and 243. At 15 the suppression was weak, at 243 rather strong, as judged by relative growth on glucose minimal medium (see line 2 o f table II). No suppression was seen with UAA243 or UAG243 (UAA and U A G were not tested at position 15). The barlO1 plasmid and the 'no insert' plasmid exhibited ~o suppression whatsoever. We also observed a synergistic effect of combining b a r + and AC1054. AC1054 is a helix 34 mutant o f 16S rRNA that has been demonstrated to be involved in peptide chain termination [11-14]. As can be seen in table II, the combination o f b a r + and AC1054 results in enhancement o f b a r + suppression at position 15 and enhancement o f AC1054 suppression at 211. No change was seen with U A A or UAG at 211. Suppression o f UGA243 was too strong with either bar+ or AC1054 to see a synergistic effect under the conditions used. Finally b a r l O 1 had no effect on AC1054 with U G A (table II) or with UAA and UAG at positions 211 and 243 (again, UAA and UAG at position 15 were not

pairing with the 1109-1205 region (in helix 34) o f 16S rRNA. This region, the helix in general [11, 12, 17, 18] and that nucleotide sequence in particular [12-14], is known to be involved in a codon-specific way in termination at U G A codons. We imagine that the association of Bar R N A with helix 34 and its consequent base-pairing prevents the 1199-1205 region o f 16S rRNA from achieving either the proposed base-pairing with a U G A codon in m R N A [11-14] or some other necessary interaction, thereby preventing the triggering o f the subsequent steps in the termination process. In Pth + (Rap +) cells, the basepairing with 16S rRNA is not effective enough to prevent termination so extensively as to be lethal for the cells or even to inhibit 2~ growth. In Pth- cells its competitiveness is somehow enhanced, presumably due to an existing defect in termination. We imagined, however, that even in Pth + cells, Bar RNA can basepair to some extent with 16S rRNA. u.~ suggested, therefore, that if a sensitive-enough test existed, we might be able to see evidence o f that interference with termination. The precise test existed, namely, suppression o f nonsense mutations. A major aspect o f the bar sequence was the presence o f UC/A and the existence o f a b a r mutation resulting from a G to A change in the middle o f the triplet. Consistent with the proposed interaction with the UGA-specific sequence of 16S rRNA, we predicted that the cloned bar+ product would be able to suppress some U G A mutations but not UAA or U A G and that the b a t - mutant product would be seen not to suppress those U G A mutations. Finally, it was reasonable to suggest that in some instances, Bar + R N A would suppress U G A ~znoroi=tio~lhz

uzlth

a h~l;~

"IA

m . . n. .

t u~ =t n,t.

1575

tLv,~ ~ ool -[.q,~u ~ A ) t] .

1 ~~ . . ~ . t=- lxl .~-' l,~. -T~A- .

When examining trpA mutant strains for suppression (growth on glucose minimal medium) we always included plates that did not require suppression (glucose minimal with either indole or trypto-

To test these three aspects o f the first prediction, we had at our disposal a high-copy number plasmid in three forms, with the b a r + region, with a b a r - region ( b a r l O l ) and with no inserted bat" region ('no insert

Table 1I. Synergistic effects of ~. bat "+and r r s B ( S u U G A - A C I 0 5 4 ) a on UGA termination. Suppression oftrpA(UGA)

Genes tested 15 h

211 b

AC 1054 a

._c

+c

bar+a barlOl d

+/.

-

AC ! 054 + bar+ AC 1054 + barlOl

.

23~ h

-

_

.

+

++

-

+

243 h

++

++

.

nte nte

++ ++

aAC1054 is short-hand for rrsB(SuUGA-AC1054). The latter designates a specific UGA-suppressing mutant form of the gene for 16S rRNA. In the strains tested, AC1054 was present in the chromosome, bUGA codon positions in trpA. c+ and - design~ate growth on glucose minimal medium. +/- and ++ designate degrees of growth that were clearly distinguishable from +. u nne wild-t e and mutant bar se uences (bar + and barlOl) were present in a multicopy plasmid derived from pBR322 [4]. We also testedYtPe plasmid without :a~y bar insert. The results with that plasmid were the same as those with the barlOl plasmid, eNot tested.

1576

EJ Murgola, G Guarneros ture sensitive for growth, as expected for pth(ts) mutants, but each parent strain, although Trp- at all temperatures, grows well at 43°C (results not shown). On glucose minimal medium without Trp, we observed a coincidence o f temperature sensitivity and prototrophy for Trp, suggesting that pth(ts) was involved in suppression o f nonsense mutations. At low temperature (30°C), all three pth(ts) trpA mutants are Trp+. Besides the coincidence o f the ts and Trp ÷ phenotypes, we note the temperature conditional nature o f the suppression o f the trpA mutations (table III). That is. although the growth of the suppressed mutants in Trp minimal is not affected by temperatures up to 40°C, the suppression (growth on minimal) is clearly decreased as the temperature is increased from 34 to 37°C and then from 37 to 40°C. As a further attempt to observe the co-variance o f temperature sensitive growth and suppression of trpA nonsense mutations, we selected for temperature resistant revertants o f 9A and 33A at high temperature. (The term 'revertant' here is used in the broadest sense; it includes not only primary site changes and intragenic second-site changes but also extragenic revertants, that is 'suppressors'.) After purification, these revertants were tested for temperature resistance and trpA suppression. The question here is whether a single step reversional event simultaneously affects trpA suppression while, o f course, affecting temperature sensitivity. The answer was yes. In fact, the revertants fell into different classes according to degree o f resistance to high temperature and suppression o f the trpA mutations. The revertants have yet to be mapped. In particular, we do not know whether any is o f the G i 0 2 type isolated several years ago by Anderson and Menninger [20].

phan added) to assess any effects o f the particular genotypic construct on general growth. In this way, we noticed that the combination of AC1054 with the bar + plasmid (but not with the barlOl plasmid or the "no insert' plasmid) resulted in decreased general growth. From this observation, we can suggest that this combination should inhibit ~. vegetative growth, analogous to the situation with ~. bar ~ in a Pth- cell.

The second prediction The basis for the second prediction was the reasoning that, if a Pth defect is required to see either o f the two bar + effects, cell lethality and ~. growth inhibition, and if the b a r + effects could be due primarily to a direct effect on peptide chain termination, then Pth must be involved somehow in chain termination. Furthermore, if the latter is correct, then we might be able to see evidence of the involvement o f Pth in termination by observing suppression o f nonsense mutations by a defective Pth. such as by pth(ts) at some temperature lower than the lethal temperature. Finally, if such suppression is observable, it should be possible to demonstrate suppression o f at least one mutation involving each o f the three termination codons. That is. we imagine that a defect in hydrolysis should be codon-nonspecific. To test this prediction, we transferred pth(ts) into strains containing trpA mutations representing each o f the three termination codons. The transfers were done with phage P1 transduction by co-transduction with zch-2410::Tn10, from strain HO300 [19]. The three best studied strains that we constructed, each containing pth(ts), were 9A, obtained with K L 2 6 5 0 (UAG243), I l A , from KL2651 (UAA243), and 33A from KL2662 (IT~Agqd.) A s ; n 4-,~hl~ ~ t¢~notes d and e), 9A, l l A , and 33A are high tempera--

•

.

.

.

.

.

.

.

.

~

n n t . r l z z v L ~ u

aat

LEIUI~.,

~IIkTUL

-

Table III. Relative g r o w t h a ofpth(ts)-containing trpA nonsense mutants on glucose minimal medium.

Growth temperature (°C)

trpA strainsb

9A(UAG243) KL2650 I 1A(UAA243 ) KL2651 33A(UGA234) KL2662

30

34

+++c _c +4 + . +++ .

+++ . +++ .

37 ++ .

. .

.

.

+++ .

43

+

_c.d e _a e _d e

.

++ .

40

+ .

++

+ .

aRelative growth was roughly estimated using patch growth on agar plates [25, 26] containing glucose minimal medium with or without 20 l.tg/ml of L-tryptophan. The patch replica plates, at all 5 temperatures, were examined each day for several days. bFor relevant genotype and construction details, see text. c_ designates no growth on glucose minimal medium. +, ++ and +++ designate three degrees of growth. To properly estimate relative suppression, the growth on glucose minimal (requires suppression) was normalized, at each temperature, to the growth on glucose minimal plus Trp. As it turned out, growth of 9A, 11A and 33A and Trp minimal was not affected (relative to the temperature-resistant parent strains) by higher temperatures (except 43°C) even up to 40°C. dAt 43°C, even the Trp-supplemented plates did not grow. eAt 43°C, the Trp-supplemented plates grew only slightly less well that at 40°C.

Model for lambda Bar RNA inhibition Reflections and p r o j e c t i o n s We have proposed a peptide chain termination model for the inhibitory actions o f 3, bar, the primary premise o f which is that Bar R N A can interfere with normal UGA-dependent chain termination by basepairing with nucleotides in the 1199-1205 region of 16S rRNA. The model explains the data accumulated to date and leads to testable predictions. Preliminary evidence supporting two basic predictions has been presented, namely, that: a) Bar + RNA leads to UGAspecific suppression in the absence o f a Pth defect, and suppresses UGA synergistically with AC1054, a mutant 16S r R N A known to affect U G A termination; and b) p t h ( t s ) , initially implicated in termination only by its required association with b a r inhibition, is involved in the ability to suppress all three types o f nonsense mutation. We have also predicted that the combination of AC1054 with the b a r + plasmid will cause ~ inhibition, an effect that should not be seen with the b a r l O 1 plasmid or with just AC1054 by itself. I f this result is obtained, it will bring us full circle, connecting the b a r + plasmid lethality with phage inhibition by ~, b a r and placing both b a r phenomena comfortably in the lap o f peptide chain termination. Furthermore, the ability o f the combination o f ACI054 and the b a r ~ plasmid to satisfy, so to speak, the requirement for a Pth defect, will implicate Pth even more rigorously in peptide chain termination. A direct demonstration o f the base-pairing interaction between Bar RNA and 16S rRNA should be achievable using the ap.propriate complementary, compensating changes m the genes for both ~,,r A~ ~,l~,uiwm~, 4, . . . . . . . . . . . . . t . c im pli cation o~ . . Pth . .in . ~:nam ~,~-~,~. termination raises a number o f interesting questions concerning the manner o f its involvement. Is Pth the hydrolase for ribosome bound ptRNAs? Is it part o f the hydrolase? Does it interact with any o f the ribosomal RNAs, in particular with the peptidyl-transferase center o f the large ribosomal subunit? Does Pth interact with peptide chain release factors (RFs)? In particular, does RF3 have anything to do with any of this? The molecular weight o f RF3 has been estimated at roughly 44 000 Da [21] while Pth is roughly half that [5]. So, for example, perhaps RF3 is a dimer o f Pth or a dimer of Pth and the ribosome-releasing factor (RRF) which has a molecular weight o f about 22 000 Da [22]. Alternatively, maybe Pth combines with RF3 to form a complex o f non-identical subunits, a heterodimer that performs the hydrolysis of ribosome-associated ptRNAs. It would be fitting if RF3 forms a mechanistic 'bridge' between the actions o f RF1 and RF2, which it enhances, and the hydrolysis o f ptRNA. Finally, even if RF3 is not involved in hydrolysis, perhaps Pth interacts with RRF to achieve polypeptide release.

! 577

It is possible to propose ahernative explanations of the inhibitory phenomena, particularly for the involvement of Pth. One could imagine, for example, that a defective Pth produces a problem at initiation. How does this relate to the anti-termination action o f Bar RNA? As mentioned above, E has 13 pairs of genes with overlapping termination and initiation signals (AUGA). Although E coli seems not to have many o f this type o f overlap, it could be that the initiation and termination o f a given mRNA are somehow connected. As for a possible molecular medium for this connection, a feature of 16S rRNA that is involved in termination (as discussed above) includes also the potential for involvement in initiation. The 1199 to 1205 sequence, 3'-UACUACU-5', contains two tandem triplets (3'-UAC-5') complementary to AUG and also includes two 'frames', if you will, of tetranucleotides complementary to AUGA. However, one would have to try to explain the involvement of Pthin nonsense suppression. Furthermore, there seems to be no compelling evidence for this more complicated scenario. Consequently, at the moment we find our model particularly attractive and useful. It leads to further testable predictions that we are now pursuing. Meanwhile, if it is correct, Bar RNA cat~ be viewed as a quasi antisense RNA [23]. As such it: a) exemplifies a minimal homology requirement for the interaction (three nucleotides; although some secondary structure feature is likely to be necessary), not unlike the association o f two tRNAs with complementary anticodons [24]; and b) points to a novel kind o f target for antisense R N A action, not the genome or mRNAs of a biological insult such as a virus but the translation apparatus, by way o f rRNA, of the host cell. Why, however, the site for such an antisense RNA should have evolved as a stable part o f the ~, genome is a most intriguing question.

Acknowledgments We are greatly indebted to Kathy Hijazi (Houston lab) for performing most of the experiments that provide the initial demonstration of the validity of the basic predictions of the termination model of ~. bat" inhibition in Pth- cells. EJM is grateful to Ai Dahlberg for discussions of possible rRNA involvements in initiation and termination of polypeptide synthesis. The work done for this report was supported in part by grant GM21499 to EJM from the National Institute of General Medical Sciences, USA and by grant No 891573 from CONACYT, Mexico and by an International Research Scholars Award from the Howard Hughes Medical institute to GG.

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EJ Murgola, G Guarneros

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