Limitation of Ribosomal Protein L11 Availability in vivo Affects Translation Termination

doi:10.1016/S0022-2836(02)00304-2 available online at http://www.idealibrary.com on

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J. Mol. Biol. (2002) 319, 329–339

Limitation of Ribosomal Protein L11 Availability in vivo Affects Translation Termination Natalya Van Dyke†, Wenbing Xu† and Emanuel J. Murgola* Department of Molecular Genetics, Box 11 The University of Texas M. D. Anderson Cancer Center 1515 Holcombe Boulevard Houston, TX 77030-4009 USA

Historically referred to as “the GTPase center”, the L11 binding region (L11BR) of Escherichia coli 23 S rRNA is a highly conserved structure that has been implicated in several essential functions during protein synthesis. Here, in vivo expression of an RNA fragment containing that structure was found to affect translation termination in a codon-specific manner. The cause of these effects appeared to be titration of ribosomal protein L11, since normal phenotypes could be restored by simultaneous overproduction of wild-type L11 but not mutant L11. Subsequently, altered termination phenotypes were produced when the availability of L11 was limited by overexpression of RNA antisense to L11 mRNA and, finally, by inactivation of the chromosomal L11 gene, and they too were reversible by simultaneous expression of cloned L11. Our results indicate that in the intact cell the L11BR is an integral functional unit important for translation termination and that the presence of L11 in ribosomes is required for UAG-dependent termination and is somewhat inhibitory of UGA-dependent termination. q 2002 Elsevier Science Ltd. All rights reserved

*Corresponding author

Keywords: 23 S rRNA fragment; ribosomal protein L11 binding region; chromosomal rplK gene knockout; translation termination; nonsense suppression

Introduction The highly conserved structure comprising nucleotides 1051– 1108 of Escherichia coli 23 S rRNA (Figure 1) is the binding region for ribosomal protein L11.1,2 Historically referred to as “the GTPase center”, it has been found to be essential for several steps in protein synthesis.3 – 7 This L11 binding region (L11BR) is an elongation factor G-binding site8 – 10 that is required for proper elongation factor function.11,12 It has also been shown to be the target site for a family of thiazole antibiotics including thiostrepton and micrococcin.13 – 17 Furthermore, hydroxyl radical mapping indicated that positions 1072 –1074 and 1093 –1095 of the L11BR rRNA are in the immediate environment of bacterial release factor 1 (RF1) bound to the ribosome.18 Finally, it appears that L11BR RNA and ribosomal protein L11 play a crucial role in the termination of protein synthesis.19 – 23 † N.V. and W.X. are joint first authors. Present address: W. Xu, P.O. Box 382009, Cambridge, MA 02238, USA. Abbreviations used: L11BR, L11 binding region; RF, release factor. E-mail address of the corresponding author: [email protected]

Analysis of small parts of the ribosome in vitro has proven very successful in enriching our understanding of ribosomal structure and function.8,24 – 28 A recent in vivo approach for studying rRNA sites involved in translation termination is based on screening of random rRNA fragments to identify those E. coli 23 S rRNA fragments that cause the suppression of nonsense mutations.29,30 Although precise mechanisms of UGA readthrough by those fragments remain to be elucidated, it seemed likely that the selective expression of rRNA fragments corresponding to segments that have been implicated in translation termination by other studies would nevertheless contribute to the elucidation of ribosomal functional interactions and mechanisms. In the study reported here, we investigated the involvement of the L11BR of 23 S rRNA in the termination of protein synthesis in vivo by selectively expressing a defined RNA fragment containing the L11BR segment of 23 S rRNA. We found that the in vivo expression of that fragment caused a decreased level of termination at UAG stop codons and an increased level of termination at UGA stop codons. These changes in translation termination could be reversed by the concomitant expression of the ribosomal protein L11. Similar results were obtained when the synthesis of L11

0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

330

L11 Binding Region of 23 S rRNA in Termination

Figure 1. (a) Representation of the secondary structure of 23 S rRNA. The L11 binding region (L11BR)-containing segment that was cloned (nt 1030 –1124) is shown in boldface. (b) Sequence and secondary structure of the cloned, L11BR-containing segment of 23 S rRNA (nt 1030–1124).

was decreased or eliminated by overexpression of RNA antisense to L11 mRNA and by inactivation of the chromosomal L11 gene. Our findings suggest that RF1 interacts with protein L11 and release factor 2 (RF2) interacts with the RNA of the L11BR.

Results In vivo expression of a 23 S rRNA gene fragment To examine the functional role of the 23 S rRNA L11BR in termination of protein synthesis, we over-

expressed in vivo an RNA fragment containing the L11BR. To do this, we cloned a 95 base-pair DNA fragment corresponding to nucleotides 1030 –1124 (Figure 1(b)) into the expression vector p5S (Table 1), under the transcriptional control of the PL promoter. Expression from the resulting plasmid, pGTP (Table 1), produced a 131 nt transcript containing the 95 nt rRNA segment upstream of a 36 nt plasmid-derived sequence. To verify the expression of this transcript in vivo, we did a Northern blot analysis of total bacterial RNA. As shown in Figure 2, the 131 nt transcript containing the 23 S rRNA L11BR was clearly detected in RNA from cells transformed with the expression

331

L11 Binding Region of 23 S rRNA in Termination

Table 1. Plasmids used in this study Plasmids

Relevant characteristics

pCAT101 pCAT103 pDCAT p5S

pACYCl84 derivative containing the cat gene with a UGA nonsense mutation at codon 74 pACYC184 derivative containing the cat gene with a UAG nonsense mutation at codon 74 pACYC184 derivative with deletion of its 412 nt Pvu II segment pNO2680 derivative with deletion of 16 S and 23 S rRNA genes, but still containing the 5 S rRNA gene p5S derivative containing the L11BR sequence p5S derivative with deletion of 84 nt from the 50 end of the 5 S rRNA gene

pGTP pD5S pPOT1 pAntF pAntN pAntC pL11 pL11Ala130 pL11Ala130/Val131 pDL11

pPOT1 expressing full-length RNA antisense to L11 mRNA pPOT1 expressing RNA antisense to the N-terminal part of L11 mRNA pPOT1 expressing RNA antisense to the C-terminal part of L11 mRNA pDCAT expressing wild-type L11 pDCAT expressing the L11 mutant Gly130Ala pDCAT expressing the L11 double mutant Gly130Ala/Thr131Val pDCAT carrying L11 gene with 83 codon deletion

plasmid pGTP. Comparison with endogenous levels of bacterial 23 S rRNA indicated that the 131 nt transcript was present at a fivefold excess over that of the full-length large ribosomal RNA. This molar excess would thus allow the plasmidderived L11BR fragment to serve as an effective competitor for specific RNA-binding proteins in vivo.

Suppression of UAG nonsense mutations by overexpression of the L11BR rRNA fragment We were interested here in determining whether expression of the L11BR rRNA fragment affects termination of protein synthesis. As a model system,

Figure 2. Detection of the in vivo expression of the plasmid-encoded 23 S rRNA L11BR fragment by Northern blot analysis. Lane 1: RNA from E. coli cells transformed with the p5S plasmid. Lane 2: RNA from E. coli cells transformed with the pGTP plasmid. Total RNA was extracted from E. coli cells and hybridized with 32P-labelled oligonucleotide complementary to the L11BR region of 23 S rRNA.

Reference or source 30 30 This work 39 This work This work 34 This work This work This work This work This work This work This work

we tested the fragment-mediated suppression of nonsense mutations in the trpA gene. Plasmids pGTP, p5S, and pD5S (Table 1) were introduced individually into bacterial cells harboring UGA or UAG nonsense codon mutations at each of several trpA codon positions (15, 115, 211, 234, and 243). The transformants were tested for the ability to grow on glucose minimal medium lacking tryptophan. As shown in Figure 3, expression of the L11BR rRNA fragment caused readthrough of the UAG nonsense mutation at position 243. Suppression of UGA243 was not observed, nor of either UAG or UGA at positions 15, 115, 211, and 234 (data not shown). Suppression of trpA(UAG243) was not the result of an overexpression of nonspecific RNA fragments, since overexpression of related control RNAs (either bacterial 5 S rRNA from plasmid p5S or the 36 nucleotides from the 30 end of 5 S rRNA from plasmid pD5S) did not cause suppression of that nonsense mutation. These results demonstrate that overexpression of the L11BR rRNA fragment causes suppression of the UAG nonsense mutation at codon position 243 of trpA. To test for L11BR fragment-mediated UAG suppression with a different reporter gene, we used a cat gene containing a UAG termination codon at position 74. Cells carrying the CAT reporter gene containing either the UAG nonsense mutation (pCAT103) or an internal deletion (pDCAT) were transformed with the L11BR fragment-encoding plasmid (pGTP) or with the p5S or pD5S control plasmids. Transformants were tested for their ability to grow in LB medium containing chloramphenicol (25 mg/ml). The results of this experiment are shown in Figure 4(a). pCAT103 cells transformed with either of the control plasmids, p5S and pD5S, remained sensitive to chloramphenicol, indicating the lack of suppression of the UAG nonsense mutation. Only the pCAT103 cells transformed with the L11BR fragment-expressing plasmid pGTP grew very well under these conditions, indicating that the RNA fragment efficiently suppressed the UAG nonsense mutation.

332

L11 Binding Region of 23 S rRNA in Termination

Figure 3. Readthrough of a UAG nonsense mutation caused by the in vivo expression of the 23 S rRNA L11BR fragment. E. coli cells possessing a UAG nonsense mutation at codon position 243 of the trpA gene were transformed with either p5S, pD5S, or pGTP. After single-colony isolation, cells were streaked on glucose minimal plates plus ampicillin, supplemented or not with indole.

Figure 4. In vivo expression of the L11BR fragment of 23 S rRNA has opposite effects on UAG-and UGA-dependent termination of protein synthesis. (a) Suppression of a UAG nonsense mutation caused by the in vivo expression of the L11BR fragment. E. coli cells carrying either the pCAT103 plasmid containing a cat gene with a UAG nonsense mutation or the pDCAT control plasmid were transformed with either pGTP, which encodes the suppressor RNA fragment, or p5S or pD5S, which express either wild-type 5 S rRNA or a 30 fragment, respectively. After single-colony isolation, cells were streaked on LB agar medium containing ampicillin (Amp) and tetracycline (Tet), with or without 25 mg/ml chloramphenicol (Cam) as indicated. (b) Enhancement of UGA-dependent termination by expression of the L11BR fragment. E. coli cells carrying either the pDCAT control plasmid or the pCAT101 plasmid, which contains the cat gene with a UGA nonsense mutation, were transformed with either pGTP, p5S, or pD5S. After single-colony isolation, transformed cells were streaked on LB agar medium containing ampicillin (Amp) and tetracycline (Tet), either with or without a low concentration (10 mg/ml) of chloramphenicol (Cam) as indicated.

333

L11 Binding Region of 23 S rRNA in Termination

This effect was not due to the non-specific inhibition of chloramphenicol sensitivity. The pDCAT cells transformed with pGTP exhibited no growth, indicating the requirement for a suppressible cat mutation. Thus in vivo expression of the L11BR-containing rRNA fragment promotes UAG suppression. When the same experiment was done with a CAT gene having UGA at the same codon position, no suppression was observed with any of the plasmids (data not shown).

Increase of UGA-dependent termination by overexpression of the L11BR rRNA fragment While the expression of the 23 S rRNA L11BR fragment in vivo leads to suppression of UAG, but not UGA, nonsense mutations, it was of interest to determine whether it affects UGA-dependent termination in a reciprocal way. Previously it was found that the UGA termination codon at position 74 of cat (pCAT101) was somewhat leaky.30 Cells with this reporter gene allele are known to be resistant to low concentrations of chloramphenicol. To test the effect of L11BR rRNA expression on UGA termination, the L11BR-encoding plasmid pGTP or the control plasmids p5S and pD5S were introduced individually into cells containing either the reporter plasmid pCAT101 or the control plasmid pDCAT. Resulting transformants were tested for their ability to grow in LB medium in the presence of a low concentration of chloramphenicol (10 mg/ml). As shown in Figure 4(b), pCAT101 cells transformed with either of the control RNA-expressing plasmids, p5S and pD5S, were resistant to the low concentration of chloramphenicol, as expected. However, transformants containing both pCAT101 and pGTP surprisingly exhibited significant sensitivity under these conditions. Such is not due to a non-specific decrease in cell viability, since both pGTP transformants grew well in the absence of chloramphenicol (left panel). This suggests that expression of the L11BR rRNA fragment in vivo promotes UGA-dependent termination. The UGA termination-enhancing effect of overexpression of the L11BR RNA was confirmed by observing antisuppression of UGA nonsense mutations in the presence of tRNA- or rRNA-suppressors of UGA mutations. In particular, pGTP was introduced into strains containing trpA (UGA211) and either a glycine tRNA UGA suppressor or a lysine tRNA UGA suppressor as well as into a strain containing trpA(UGA115) and the 16 S rRNA UGA suppressor C1054A.19,22,23 In all three cases, expression of the L11BR RNA dramatically decreased the suppression of the trpA mutation (data not shown). The results suggest that the antisuppression of UGA nonsense mutations by L11BR overexpression is a general phenomenon caused by enhancement of UGA-dependent termination.

L11BR rRNA fragment-mediated changes in translation termination involve the large subunit ribosomal protein L11 We hypothesized that the overexpressed L11BR fragment RNA may actually titrate L11, thereby generating some L11-deficient ribosomes in vivo. Therefore, to test whether the previously observed termination defects were the result of an L11 deficiency, we examined termination codon readthrough in vivo in the presence of L11 protein co-overexpressed with the L11BR rRNA fragment. As controls we overproduced instead the singly mutant L11 protein L11Gly130Ala (pL11Ala130) and the doubly mutant protein L11Gly130Ala/ Thr131Val (pL11Ala130/Val131), as well as a deletion mutant of L11 (pDL11). Previously it had been shown that the Gly131Ala and Thr132Val mutations dramatically reduced L11 binding to 23 S rRNA in vitro.31 The results of our co-transformation experiments are presented in Table 2. The UAG-suppression effect caused by the overexpression of the cloned L11BR rRNA fragment was completely reversed by simultaneous overexpression of a plasmid-derived, wild-type L11 protein, but not by mutant L11. The dramatic nature of this effect was most apparent from the significant reduction of UAG suppression seen with just the very low level of wild-type L11 protein expression present in the absence of IPTG induction. Similarly, the partial reduction of suppression by the L11Ala131 mutant protein might be ascribed to the very high levels of mutant protein synthesized following IPTG induction.

In vivo expression of L11 antisense RNA has the same effect on UAG-dependent termination as L11BR rRNA fragment overexpression Defective UAG-dependent termination can also be achieved by an entirely different approach to Table 2. Reversal of L11BR RNA-induced nonsense suppression by overproduction of wild-type protein L11

Plasmid names pGTP, pGTP, pGTP, pGTP, pGTP,

pACYC184 pL11 pL11Ala130 pL11Ala130/Val131 pDL11

Suppression of trpA(UAG243) 2IPTG

þ IPTG

þþþ þ þþþ þþþ þþþ

þþþ 2 þþ þþþ þþþ

Cell growth was monitored at 35 8C. The growth of replicaplated patches of cells on agar plates with glucose minimal medium containing ampicillin (100 mg/ml), tetracycline (10 mg/ml), with or without IPTG (1 mM). No differences were observed in cell growth on glucose minimal medium containing ampicillin (100 mg/ml), tetracycline (20 mg/ml) and supplemented with indole, either with or without 1 mM IPTG (data not shown). (2), No growth after six days; (þ to þþþ), increasing amounts of growth compared with pGTP, pL11 cotransformants.

334

L11 Binding Region of 23 S rRNA in Termination

Table 3. Effect of antisense L11 expression on suppression of trpA(UAG243) and on cell growth at 37 8C Growth on glucose minimal mediuma

Plasmid

þ Indole

2Indole

pAntF pAntN pAntC pPOT1

Growth on LB mediumb

2IPTG

þ IPTG

2IPTG

þIPTG

2IPTG

þ IPTG

2 2 2 2

þ þ 2 2

þþþþ þþþþ þþþþ þþþþ

þþþ þþþ þþþþ þþþþ

þþþþ þþþþ þþþþ þþþþ

2 2 þþþþ þþþþ

a In the test for suppression of UAG nonsense mutations, E. coli cells with a UAG nonsense mutation at codon position 243 in the trpA gene were transformed with plasmids encoding antisense RNAs to all or part of the L11 mRNA (pAntF, pAntN, pAntC) or with the control vector pPOT1. Cells were replica-plated onto agar plates containing glucose minimal medium and 100 mg/ml ampicillin, with or without indole or 1 mM IPTG as indicated. The suppression activity was examined at 31, 35, 37, and 41 8C. Suppression activity was not detected at 31 8C and 35 8C. b Effect of expression of antisense RNAs to different parts of the L11 mRNA on cell growth in rich medium (LB) was monitored at 31, 35, 37, and 41 8C. No effects on growth were observed at 31 8C and 35 8C. (2), No growth after six days; (þ to þþþþ), increasing amount of growth compared with control.

limiting the availability of L11 for ribosome assembly, namely by overproduction of RNA antisense to L11 mRNA in vivo, which is aimed at decreasing the synthesis of the L11 protein. Plasmids encoding antisense transcripts corresponding to different regions of L11 mRNA were constructed (Table 1). These included antisense to the entire L11 mRNA (pAntF), or to the N-terminal (pAntN) or C-terminal (pAntC) portions of L11 mRNA. The corresponding parent expression vector (pPOT1) was used as a control. The expression of antisense transcripts to L11 mRNA was tested for effects on cell growth and for suppression of UAG mutations. Cells expressing antisense to the entire L11 mRNA and to the N-terminal portion exhibited high temperatureconditional lethality (37 8C and 41 8C) in rich medium (Table 3). In contrast, cells were able to grow, albeit slowly, at high temperature on supplemented minimal medium (i.e. “ þ Indole”). For the examination of nonsense suppression, bacterial strains with stop codons in the trpA gene at codon positions 15, 115, 211, and 243 were transformed with pAntF, pAntN, pAntC, or the control pPOT1. Each of the resulting strains was examined for an IPTG-dependent Trpþ phenotype. IPTG-dependent suppression of UAG at codon position 243 in trpA was observed for the cells containing the antisense to complete L11 mRNA (pAntF) and to the N-terminal part of L11 mRNA (pAntN) (Table 3). Finally, to determine whether UAG suppression produced by the expression of an antisense transcript was dependent on the level of expression of L11, we examined the expression of antisense to L11 mRNA in the presence of overproduced L11 protein. The simultaneous expression of L11 completely reversed the antisense-induced high temperature-conditional inhibition of cell growth and the UAG suppression (data not shown). Suppression of UAG nonsense mutations caused by the absence of protein L11 To completely eliminate synthesis of ribosomal protein L11, we constructed a strain having a dis-

rupted chromosomal rplK, the gene that codes for L11. E. coli strains that lost the ability to express L11 were found previously after selection for reversion of erythromycin dependence.32 However, besides the apparent absence of L11, some of those strains had alterations in the expression of other ribosomal proteins. So, in addition to the lack of understanding of the connection between mutation to erythromycin independence and the absence of L11, it is possible that multiple changes in the ribosomes occurred in these strains. Meanwhile, a number of new methods for the disruption of procaryotic open reading frames were recently described. In particular, the gene replacement protocol of Link et al.33 offers the advantage of making precisely engineered changes in only the target gene. Therefore we constructed a strain with a chromosomal rplK gene knockout using this protocol (see Materials and Methods). Various derivatives of that strain were made by introducing specific plasmids (Table 4). The strains also contained a UAG nonsense mutation in the trpA gene (codon position 243), which allowed the testing of UAG readthrough. The phenotypes characteristic of an inactivated L11 gene are illustrated in Figure 5. Strain NDV004 (Table 4), which has the uncomplemented rplK knockout, did not survive at high temperature, but at low temperature exhibited suppression of trpA(UAG243), that is, growth on glucose minimal medium without indole or Trp. Both phenotypes, however, were reversed when the rplK knockout was complemented by the expression of plasmid-derived L11 protein (strain

Table 4. rplK-Knockout strains constructed for this study Strains NDV003 NDV004 NDV006 NDV007 a

See Table 1.

Relevant genotypes DrplK/pL11a/F0 trpA(UAG243) DrplK/pDCATa/F0 trpA(UAG243) DrplK/pCAT101a (UGA74)/F0 trpA(UAG243) DrplK/pCAT103a (UAG74)/F0 trpA(UAG243)

335

L11 Binding Region of 23 S rRNA in Termination

Figure 5. UAG suppression and high-temperature lethality of cells containing an inactivated rplK gene. To test for suppression of the UAG nonsense mutation at codon position 243 in the trpA gene, strains NVD003 and NVD004 (Table 4) were streaked on glucose minimal medium containing 10 mg/ml tetracycline and 1 mM IPTG and incubated at 31 8C. Suppression-independent growth was monitored at 31 8C on glucose minimal medium supplemented with indole, tetracycline and IPTG. Temperature-dependent growth was monitored on LB medium with tetracycline and IPTG, and incubated at 43 8C. Plates are shown after five days.

NDV003). To examine the codon specificity of the readthrough caused by the absence of L11, plasmids pCAT101 and pCAT103 (Table 1) were introduced independently into the rplK knockout strain, creating strains NDV006 and NDV007, respectively. Strain NDV004 contained the control plasmid pDCAT. The three strains were tested for their ability to grow in LB medium in the presence of various concentrations of chloramphenicol (Table 5). The rplK chromosomal knockout strain carrying the plasmid (pCAT103) encoding the cat gene with a UAG nonsense mutation at codon position 74 (strain NDV007) exhibited significant growth in the presence of high levels of chloramphenicol, indicating that cells without protein L11 efficiently suppress the UAG mutation. The

Table 5. Effect of a chromosomal L11 gene (rplK ) knockout on the suppression of cat gene nonsense mutations

Strainsa NVD004 (DCAT) NVD006 (UGA74) NVD007 (UAG74)

Growthb on LB medium with different concentrations (mg/ml) of chloramphenicol 0

10

20

30

40

50

1.5 1.5 1.5

2 2 1.5

2 2 1.5

2 2 1.5

2 2 1.5

2 2 1.5

a The strains (see Table 1) lacked the L11 protein because of a chromosomal deletion within the rplK gene. They each carried a plasmid that contains a cat reporter gene with either a UAG or UGA nonsense mutation at position 74 or a deletion of the cat gene. b Suppression of nonsense mutations was tested by monitoring cell growth in LB medium with different concentrations of chloramphenicol. Average colony diameters (mm) were determined after five days incubation at 31 8C on LB plates containing the indicated concentrations of chloramphenicol. (2), No colonies formed.

UAG suppression was not due to the non-specific inhibition of chloramphenicol sensitivity, since the rplK knockout strains containing either the UGA mutant cat gene (pCAT101) or the control plasmid (pDCAT) did not grow in the presence of any of the tested concentrations of chloramphenicol.

Discussion Prompted by earlier findings of mutations in the L11BR (previously referred to as “the GTPase center”) that affected translation termination at UGA19,21 – 23 and by the interesting results of screening a library of rRNA gene fragments34 for nonsense suppression,29,30 we asked whether in vivo expression of an RNA fragment containing the L11BR had an effect on translation termination. We observed that expression of the RNA fragment in intact cells affected translation termination in a codon-specific manner, decreasing termination at UAG (suppression of UAG nonsense mutations) and increasing termination at UGA (inhibition of UGA leakiness and suppressor tRNA- or suppressor rRNA-mediated UGA suppression). In general, we have demonstrated here that in vivo expression of a defined rRNA fragment provides a useful method for limiting the availability, in intact cells, of rRNA-interactive molecules and for thereby furthering our understanding of in vivo ribosomal mechanisms operating during termination, and likely during other stages of polypeptide synthesis. In particular, we identified the mechanism for the action of the L11BR-containing fragment in vivo. Xing et al.31 showed in vitro that the L11 protein recognizes and forms a complex with the L11BR fragment. In our in vivo experiments, the overexpressed L11BR rRNA fragment titrated the L11

336

protein, resulting in L11-deficient ribosomes defective in UAG-dependent termination but more effective in UGA-dependent termination. This mechanism was validated by the restoration of normal termination phenotypes by the overproduction of wild-type but not mutant L11 protein. In addition, limitation of L11 availability for ribosome assembly was accomplished by decreasing the synthesis of L11 through the overexpression of RNA antisense to L11 mRNA. The expression of L11 antisense in vivo resulted in UAG suppression and a high-temperature growth inhibition, both of which could be reversed by simultaneous expression of cloned L11. Although the antisense approach has been widely used in various kinds of studies, it can present problems as a method for limiting the availability of particular molecules in cells that express polycistronic mRNAs or that employ regulatory mechanisms at the translational level. Therefore, to demonstrate conclusively the involvement of L11 in termination, we created a deletion in the chromosomal rplK gene, the gene for protein L11. To do this, we used a gene replacement method designed to create precisely engineered deletions of E. coli open reading frames and to use them to replace a wild-type chromosomal gene.33 Inactivation of the chromosomal L11 gene resulted in a serious growth defect at all temperatures and lethality at 43 8C. At low temperature, the knockout strain revealed suppression of UAG mutations at codon position 243 in trpA and at position 74 of the cat gene. These phenotypes could be reversed by expression of plasmidencoded L11 protein. In bacteria, termination requires two codonspecific release factors, RF1 and RF2. RF1 works at UAA and UAG stop codons, while RF2 works at UAA and UGA.35 Thus it is reasonable to conclude that the observed UAG-specific nonsense suppression and increase in UGA-specific termination of L11-deficient ribosomes demonstrate that L11 is essential for RF1 function and has an inhibitory effect on RF2. A differential effect of L11 on the activities of the two release factors was observed previously in vitro.20 However, the classical in vitro system employed in that study did not accurately mimic the in vivo process, thus limiting the ability to draw conclusions from the data as to whether, to what extent, and precisely how the differential effect of L11 on termination comes into play in the intact cell.36 Nevertheless, as it turns out, those early in vitro results are consistent with our demonstration here of what occurs in vivo, namely that the presence of protein L11 is required for RF1 dependent termination (and is likely an RF1-interactive site on the ribosome) and weakens somewhat the functioning of RF2. Mutations in the two loops (nt 1065 – 1073 and 1093 –1098) of the L11BR 23 S rRNA cause suppression of UGA nonsense mutations.21 Both in vivo and in vitro analyses of these mutations have indicated that the loop nucleotides 1067, 1093, 1094,

L11 Binding Region of 23 S rRNA in Termination

and 1095 are RF2-interactive sites and specifically are part of the RF2 ribosome binding site.21 – 23 So initially it may have seemed somewhat surprising that overexpression of the L11BR did not affect RF2-dependent termination negatively, by titrating RF2 and thereby limiting its availability for interaction with the ribosome. However, it is known that other regions of rRNA influence termination at UGA and possibly the binding of RF2 to the ribosome, notably the nt 1054 portion of helix 34 of 16 S rRNA.19,22,23 On the other hand, the UGAspecific suppression caused by mutations in the L11BR21 indicates that RF1 does not interact directly with that rRNA structure. In our Introduction above, we noted that hydroxyl radical mapping indicated that positions 1072 – 1074 and 1093 –1095 of the L11BR rRNA are in the immediate environment of bacterial release factor RF1 bound to the ribosome.18 However, proximity under certain experimental conditions does not necessarily indicate functional interaction. Despite some structural similarity to RF2, RF1 is notably different and so must bind to the ribosome and function differently from RF2. Crystallographic analyses of isolated complexes of L11 and its rRNA binding region and cryo-electron microscopy studies of EF-G/70 S complexes have shown that the N-terminal domain of the L11 protein and the two RNA loops are situated very close to one another and capable of transient interactions.10,24 Considering those observations with the results of our functional studies (i.e. the enhancement of RF2-dependent termination with L11-deficient ribosomes), we suggest that protein L11 partly masks the rRNA loops (nt 1065 –1073 and 1093– 1098) in competition with RF2. The data and considerations presented above suggest that RF1 and RF2 interact with overlapping but distinct sites, RF1 with protein L11 and RF2 with nt 1067 and 1093– 1095 of the L11BR rRNA.

Materials and Methods Strains, plasmids, and materials Bacterial strains used for testing codon specificity of translational suppressors were derived from E. coli K-12 with different mutations in trpA. The nomenclature of trpA mutations was described previously.29,37 Extraction of plasmid DNA from bacteria and general DNA purification were performed using QIAquick series purification kits (Qiagen) following the manufacturer’s instructions. Digestion of DNA with restriction enzymes, DNA ligation, DNA amplification by PCR, and agarose gel electrophoresis were performed as described.38 DNA sequences were obtained using an Applied Biosystems Model 373A automated DNA sequencer. Plasmid construction For construction of the L11 binding region (L11BR) rRNA fragment expression plasmid, the 95 nt sequence containing the L11BR of 23 S rRNA (nucleotides

337

L11 Binding Region of 23 S rRNA in Termination

1030– 1124; Figure 1(b)) was produced by PCR amplification using the primers 50 -GACGGGGTACCCGATGTGGGAAGGCCCAGACAGC-30 and 50 -TGGTTTAGCCCCGCATGGGGAGACCCGCGCAGGCCGACTCGACCAGTG-30 (underlined are KpnI and BsaI recognition sites, respectively). This allowed addition of a KpnI restriction site upstream of the L11BR rRNA fragment and addition of a BsaI site downstream. The PCR product was digested with KpnI and BsaI, then ligated into a similarly digested p5S vector. This placed the L11BR rRNA fragment immediately downstream of the transcription start site for the PL promoter but upstream of the 5 S rRNA transcriptional stop site.39 The resulting plasmid was designated pGTP. Note that the transcript produced from this promoter is 131 nt long and comprises the 95 nt L11BR-containing segment followed, at the 30 end, by the 36 nt from the 30 terminus of 5 S rRNA. To create an equivalent control plasmid, i.e. one that expresses only the 36 nt 5 S rRNA 30 terminus, a plasmid containing an 84 base-pair deletion of the 5 S gene (pD5S) was constructed by deleting the sequences between the KpnI and BsaI restriction sites using the Expand Long Template PCR System (Roshe) and the amplimers 50 -ATGGTACCGGTCTCCCCATGCGAG-30 and 50 -CCGGTACCCGTGCTCAGTATCA-30 . This allowed the introduction of an additional KpnI restriction site immediately upstream of the 50 end of BsaI restriction site. The PCR product was digested by KpnI and ligated. The mutant fragment between the KpnI and BamHI sites was recloned into the p5S vector with the same restriction enzymes. The vector was designated pD5S. To generate plasmids for the expression of the L11 protein and for the expression of antisense L11 transcripts, the L11 gene with an additional 20 nt long segment upstream of the 50 end and 4 nt downstream from the 30 end was amplified from E. coli genomic DNA using the amplimers 50 -TAGCTAGCACCCAACTTGAGGAATTTAT-30 and 50 -TAGCTAGCTTTCTTAGTCCTCCACTACC-30 (underlined sequences are NheI sites). The oligonucleotides were designed to introduce NheI sites at both the 50 and 30 ends of the PCR product. The generated PCR fragment was digested with NheI and cloned into a similarly digested pPOT1 vector. The pPOT1 derivative carrying the L11 gene in the sense orientation was designated pPOT1L11, while the pPOT1 derivative with the reverse orientation of the L11 gene was named pAntF. A plasmid for the expression of antisense to 192 nt from the N terminus of L11 and to 20 nt upstream of the start codon was similarly constructed using the primers 50 -TAGCTAGCACCCAACTTGAGGAATTTAT30 and 50 -TAGCTAGCATTAGTCAGCGTAAACG-30 . A plasmid for the expression of the 237 nt long antisense to the C-terminal part of L11 mRNA was constructed using the primers 50 -TAGCTAGCCGTTCTTTCACTTTCGT-30 and 50 -TAGCTAGCTTTCTTAGTCCTCCACTACC-30 . These latter plasmids were named pAntN and pAntC, respectively. To create the derivative of pACYC184 for expression of the L11 protein, the KpnI fragment from pPOT1L11 containing the L11 gene was recloned into pDCAT, replacing its BsuI/SacI fragment. Plasmid pDCAT is derived from pACYC184 by deleting its 412-bp PvuII fragment. Amino acid position 130 of the L11 was mutated from Gly to Ala, by PCR site-directed mutagenesis using the pL11 plasmid as a template and the back-to-back primers 50 -CGAAGCTACTGCACGTTCCAT-30 and 50 -ATGGAGCGAGTCATCGCTTC-30 . The mutation introduced by these primers into the L11 gene

is indicated by italics. PCR-based site-directed mutagenesis was performed using an ExSite site-directed mutagenesis kit (Stratagene) following the manufacturer’s instructions. The resulting plasmid, which expresses the L11Gly130Ala mutant protein under Ptac promoter control, was named pL11Ala130. To construct a plasmid for expression of the doubly mutant L11 protein Gly130Ala/Thr131Val, the QuikChange site-directed mutagenesis kit was used (Stratagene), with PCR performed on the pL11Gly130Ala plasmid as a template, and the oligos 50 -CCATCGAAGCTGTTGCACGTTCC-30 and 50 -GGAACGTGCAACAGCTTCGATGG-30 as primers. The mutational changes introduced by these primers in the amplified L11 gene are indicated by italics. The resulting plasmid expresses the L11Gly130Ala/Thr131Val doubly mutant protein under the control of the Ptac promoter and was named pL11Ala130/Val131. Plasmid pDL11, with the codons for 83 amino acid residues (positions 40 – 122) deleted from the L11 gene, was obtained using an ExSite PCR-based site-directed mutagenesis kit (Stratagene). The PCR reaction was done with pL11 as the template and with the primers 50 GAAGCGATGACTCGCTCC-30 and 50 -GCAGAATTCCATGATGTTTAC-30 . The mutations were confirmed by automated sequence analyses using Applied Biosystems reaction protocols, and model 373A DNA sequencer, at the M. D. Anderson Cancer Center’s Automated DNA Sequencing Core Facility.

Procedure for making a chromosomal L11 gene (rplK ) knockout The L11 operon in E. coli contains genes coding for both the ribosomal proteins L11 and L1. It is known that translation of L1 does not take place unless the preceding L11 cistron is translated. Specific regions inside the L11 gene have been defined to be important for initiation and regulation of L1 translation.40 To avoid interference with translation of the L1 protein, we engineered an inframe deletion within the L11 gene that did not disrupt those regions. First, in a cloned copy of rplK, we made an internal deletion that resulted in the loss of a 249 nucleotide fragment coding for 83 amino acid residues of the L11 protein (from codon positions 40 – 122). We then replaced the wild-type chromosomal rplK gene with the cloned DrplK using essentially the gene replacement procedure described previously33 with some modifications (N.V. & E.J.M., unpublished results) Finally, we constructed various rplK-deletion strains carrying specific plasmids, for example, NVD strain numbers 003, 004, 006, and 007 (Table 4).

In vivo tests of suppression specificity Nonsense suppression specificity was tested in two reporter systems. First, E. coli strains with UGA and UAG nonsense mutations at each of five codon positions, 15, 115, 211, 234 and 243, in trpA29,37 were tested for stop codon readthrough resulting in an active trpA-encoded protein. Suppression of the nonsense mutations would be indicated by a Trpþ phenotype, that is, the ability of the cells to grow on glucose minimal medium in the absence of tryptophan or indole. Second, cells with plasmids pCAT101 and pCAT103 (Table 1) were examined for resistance to chloramphenicol.

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Analyses of RNA by Northern hybridization 38

Total RNA was prepared as described. The RNA was separated according to size by electrophoresis through a denaturing acrylamide gel and then was transferred to Zeta-Probe GT blotting membranes using a semi-dry transfer system (Bio-Rad laboratories). The RNA was located by hybridization with a radiolabeled nonadecameric deoxyoligomer complementary to nt 1032– 1050 of the L11BR-containing segment of 23 S rRNA (Figure 1(b)) according to the instruction manual of Zeta-Probe GT blotting membranes (Bio-Rad laboratories).

9.

10.

11.

12.

Acknowledgments We thank Frances Pagel for experimental assistance, Walter J. Pagel for incisive editorial comments, and Song Q. Zhao for construction of the plasmid p5S. This work was supported by grant GM21499 from the National Institute of General Medical Sciences. The automated DNA sequencing facility at the M. D. Anderson Cancer Center was supported by grant CA16672 from the National Cancer Institute.

13.

14.

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Edited by D. E. Draper (Received 6 December 2001; received in revised form 27 March 2002; accepted 28 March 2002)