The EMBO Journal vol.10 no.8 pp.2203-2214, 1991
A functional pseudoknot in 16S ribosomal RNA
Ted Powers and Harry F.Noller Sinsheimer Laboratories, University of California, Santa Cruz, Santa Cruz, CA 95064, USA Communicated by C.G.Kurland
Several lines of evidence indicate that the universally conserved 530 loop of 16S ribosomal RNA plays a crucial role in translation, related to the binding of tRNA to the ribosomal A site. Based upon limited phylogenetic sequence variation, Woese and Gutell (1989) have proposed that residues 524-526 in the 530 hairpin loop are base paired with residues 505-507 in an adjoining bulge loop, suggesting that this region of 16S rRNA folds into a pseudoknot structure. Here, we demonstrate that Watson-Crick interactions between these nucleotides are essential for ribosomal function. Moreover, we find that certain mild perturbations of the structure, for example, creation of G-U wobble pairs, generate resistance to streptomycin, an antibiotic known to interfere with the decoding process. Chemical probing of mutant ribosomes from streptomycin-resistant cells shows that the mutant ribosomes have a reduced affinity for streptomycin, even though streptomycin is thought to interact with a site on the 30S subunit that is distinct from the 530 region. Data from earlier in vitro assembly studies suggest that the pseudoknot structure is stabilized by ribosomal protein S12, mutations in which have long been known to confer streptomycin resistance and dependence. Key words: pseudoknot/ribosome/rRNA/streptomycin/ translation
Introduction The 530 loop region is one of the most highly conserved features of 16S ribosomal RNA (and, indeed, of any known RNA molecule), both in sequence and in secondary structure (Woese et al., 1975;Gutell et al., 1985). This must reflect its importance to some translational function. The information presently in hand indicates that it is involved in some aspect of A-site tRNA-ribosome interaction, i.e. the decoding process. G530 and U531 are protected from chemical probes when tRNA is bound to the 30S A site; this protection is observed even when tRNA is replaced by just its anticodon stem-loop (Moazed and Noller, 1986). Because the 530 loop has been located using several independent approaches at a site that is remote (>70 A) from the actual site of codon-anticodon interaction, it has been proposed that its tRNA-dependent protection occurs by an indirect, allosteric mechanism (Moazed and Noller, 1986; Stern et al., 1988c). Its association with A-site function is reinforced by the discovery of ochre suppression by a mutant of yeast mitochondria containing a G to A mutation at a position corresponding to G5 17 of E. coli 16S rRNA (Shen and Fox, Oxford University Press
1989), by the occurrence of streptomycin-resistance mutations in the 530 loop (Gauthier et al., 1988; Fromm et al., 1989), and by enhancement of reactivity of C525 toward dimethyl sulfate by aminoglycoside antibiotics (Moazed and Noller, 1987). Recently, we have demonstrated that point mutations involving G530 confer a dominant lethal phenotype, and that ribosomes containing such mutations are impaired in some translational step following initiation, consistent with the involvement of this strucure in A-site function (Powers and Noller, 1990). Precisely how the 530 loop might act is not at all understood, however, and is a particularly intriguing question in view of its apparent remoteness from the decoding site. Recently, Woese and Gutell (1989) have proposed an unusual higher-order structural interaction involving the 530 loop, based on comparative sequence analysis. This interaction involves base pairing between residues 524-526 in the 530 region hairpin loop with residues 505-507 in the adjoining 510 region bulge loop (Figure 1). The resulting structure is an example of the class of RNA strucures called pseudoknots (Pleij et al., 1985). These structures appear to be rare in rRNA (Gutell and Woese, 1990), and so the occurrence of a pseudoknot in such a functionally interesting context in 16S rRNA raises the possibility that it may play a functional role. Because of the extremely high conservation of primary structure in the 530 loop, sequence variation supporting the proposed structure is sparse. Furthermore, comparative analysis, although a powerful tool for analysis of RNA structure, does not inform us about the specific nature of the biological requirement for a structure. In this paper, we demonstrate directly, by site-directed mutagenesis, the requirement of this interaction for the proper functioning of the ribosome. In addition, we find that certain perturbations of the pseudoknot structure confer resistance to streptomycin, a drug that is known to interfere with the decoding process.
Results Genetic analysis of the proposed 530 stem -loop tertiary interaction The interaction involving nucleotides 505-507 and 524-526 in 16S rRNA, proposed by Woese and Gutell (1989), is shown in Figure 1.We took a genetic approach to test the existence of this interaction by reasoning that if it is essential for ribosome function, then mutations that disrupt pairing between these bases should impair cell growth, while compensatory changes that restore pairing should restore wild-type-like growth. We constructed mutations based on the sequences shown in Figure 1. As each of these variations is known to occur in two or more different eubacterial and/or mitochondrial 16S-like rRNAs, we believed they had the best potential for functioning in E. coli. Site-directed mutations were introduced into plasmid 2203
T.Powers and H.F.Noller AGCCG G 5C G-
A
GC
520-A
U A
C
CG * UA U-A
510 C-G \ C-G AAU -A U
C-G-540
GC G CGGC-
524-526 507-505
232
35
GUC CAG
3
0
ACC UGG
0
0
GCC CGG
43
GCA CGU
G
A-U C-G G-C
500
Fig. 1. (Left) Secondary structure of the 530 stem-loop region in 16S rRNA from E.coli. The tertiary interaction between positions 505-507 and 524-526, proposed by Woese and Gutell (1989), is shown in bold. (Right). Sequence variations found in two or more 16S or 16S-like rRNAs that support the proposed interaction. Differences from the E.coli sequence are shown in bold. Numbers under each phylogenetic group indicate the number of known variants. Data from Woese and Gutell (1989) and R.R.Gutell (personal communication).
pSTL102 (Triman et al., 1989), as described in Materials and methods. This plasmid contains an ampicillin-resistance gene and the entire rrnB operon from E. coli. A C to U transition at position 1192 in the 16S rRNA gene confers resistance to spectinomycin, allowing for the phenotypic characterization of plasmid-encoded 16S rRNA. E. coli strain DH1 was transformed with the mutant plasmids and the growth of these resulting strains was compared in the presence of spectinomycin (Spc) and/or ampicillin (Amp). As controls, cells were also transformed with plasmids pSTL102 and pKK3535, the latter being the Spcs parent to pSTL102. For each mutant, the amount of 16S rRNA assembled into ribosomes and polyribosomes was determined, using the U1 192 mutation to identify plasmidencoded RNA in a quantitative primer extension assay (Sigmund et al., 1988). Below we describe our results for each set of mutations examined. G506 - C525 interaction The most convincing sequence variation suggesting an interaction between the 510 and 530 regions involves pairing between G506 and C525. The sequence A506 - U525 is found within the eubacteria, in the gram-positive mycoplasmas, as well as in several different mitochondrial 16S-like rRNAs (Woese and Gutell, 1989; Figure 1). Cells carrying the U525 single mutation grew normally on Amp plates but displayed a slight reduction in colony size on Spc plates, at higher temperatures (Table I and Figure 2, pU525). Plasmid-encoded 16S rRNA from these cells was present at wild-type levels in both ribosomes and polyribosomes (data not shown). No transformants were recovered with the plasmid containing the A506 single mutation, suggesting that expression of this mutation was lethal to the host cell. We tested this directly by placing this mutation under inducible expression, utilizing the PL promoter/operator in plasmid pLK45 and the temperature-sensitive cI repressor from bactriophage lambda (Powers and Noller, 1990) (see Materials and methods). The results of induction experiments are shown in Table II. Cells transformed with plasmid pLA506 displayed a slow-growth phenotype at 420C, the induced temperature. Analysis of the distribution of plasmidencoded rRNA, using the U1 192 marker as above, showed a substantially reduced amount of mutant 16S rRNA in both ribosomes and polyribosomes (Table II). We have determined previously that the amount of plasmid rRNA
2204
incorporated into ribosomes from the PL promoter in pLK45 is substantially less (50% versus >70%, respectively) than from the natural PIP2 promoters in pSTL102 (Powers and Noller, 1990). We believe this difference in gene-dosage explains why pLK45 does not confer resistance to spectinomycin (our unpublished results). This difference may also explain why the A506 mutation is apparently lethal in pSTL102 but viable, albeit slowgrowing, under PL control. In contrast, when the A506 and U525 mutations were combined, cells grew similarly to wild-type on both Amp and Amp+Spc plates (Table I and Figure 2, pA506/U525). Plasmid-encoded 16S rRNA was also present at wild-type levels in both ribosomes and polyribosomes (data not shown). These results demonstrate that the U525 mutation can suppress the severe growth-defect caused by the A506 mutation and that the A506 mutation can suppress the weak growth-defect caused by the U525 mutation. Recently, a C to U mutation at position 525 (E. coli numbering) in tobacco chloroplast 16S rRNA has been shown to confer resistance to streptomycin (Sm) (Fromm et al., 1989). However, cells carrying pU525 failed to grow on Amp plates containing 3 mg/l Sm, the minimum concentration of Sm required to prevent the growth of cells containing wild-type plasmids (Figure 2). This result is consistent with the difficulty other groups have experienced in expressing 16S rRNA Smr mutations in E. coli (Montandon et al., 1986; Melanqon et al., 1988). This is most likely due to the fact that sensitivity to streptomycin is dominant in cells heterozygous for Smr and Sm5 alleles (Lederberg, 1951). It has been observed, however, that by combining the Ul 192 Spcr marker and a putative Smr mutation in cis, cells grow in the presence of both spectinomycin and streptomycin; spectinomycin presumably inactivates the sensitive, chromosomally-encoded ribosomes, allowing expression of the recessive streptomycin resistance (E.Morgan, personal communication). Accordingly, significant growth was observed on Amp + Spc + Sm plates by cells carrying U525, but not by cells carrying the control plasmids nor the A506/U525 double mutation (Figure 2 and Table I). Cells appearing on these plates were re-tested on selective plates and were found to maintain their original phenotype. Most importantly, no growth was observed on plates containing Sm alone, indicating that these cells had not acquired a chromosomal mutation conferring streptomycin resistance. These results indicate that resistance to
A functional pseudoknot in 16S ribosomal RNA Table I. Growth properties of 530 region pseudoknot mutants on selective platesa
Plasmid
510/530 interaction
Temperature
26°C Amp
AmpSpc
370C AmpSpcSm Amp
AmpSpc
+++
+++
-
+++
+++
+++
+++
++
+++
++
+++
+++
_
+++
+
+l-
_
+1I-
-
+++
420C Amp
AmpSpc
AmpSpcSm
+++
+++
-
++
+++
++
++
+++
-
+++
++
_
+
+
_
+
+
+
_
+1I-
-
_
+1I-
-
_
++
-
+++
++
-
+++
A functional pseudoknot in 16S ribosomal RNA
Ted Powers and Harry F.Noller Sinsheimer Laboratories, University of California, Santa Cruz, Santa Cruz, CA 95064, USA Communicated by C.G.Kurland
Several lines of evidence indicate that the universally conserved 530 loop of 16S ribosomal RNA plays a crucial role in translation, related to the binding of tRNA to the ribosomal A site. Based upon limited phylogenetic sequence variation, Woese and Gutell (1989) have proposed that residues 524-526 in the 530 hairpin loop are base paired with residues 505-507 in an adjoining bulge loop, suggesting that this region of 16S rRNA folds into a pseudoknot structure. Here, we demonstrate that Watson-Crick interactions between these nucleotides are essential for ribosomal function. Moreover, we find that certain mild perturbations of the structure, for example, creation of G-U wobble pairs, generate resistance to streptomycin, an antibiotic known to interfere with the decoding process. Chemical probing of mutant ribosomes from streptomycin-resistant cells shows that the mutant ribosomes have a reduced affinity for streptomycin, even though streptomycin is thought to interact with a site on the 30S subunit that is distinct from the 530 region. Data from earlier in vitro assembly studies suggest that the pseudoknot structure is stabilized by ribosomal protein S12, mutations in which have long been known to confer streptomycin resistance and dependence. Key words: pseudoknot/ribosome/rRNA/streptomycin/ translation
Introduction The 530 loop region is one of the most highly conserved features of 16S ribosomal RNA (and, indeed, of any known RNA molecule), both in sequence and in secondary structure (Woese et al., 1975;Gutell et al., 1985). This must reflect its importance to some translational function. The information presently in hand indicates that it is involved in some aspect of A-site tRNA-ribosome interaction, i.e. the decoding process. G530 and U531 are protected from chemical probes when tRNA is bound to the 30S A site; this protection is observed even when tRNA is replaced by just its anticodon stem-loop (Moazed and Noller, 1986). Because the 530 loop has been located using several independent approaches at a site that is remote (>70 A) from the actual site of codon-anticodon interaction, it has been proposed that its tRNA-dependent protection occurs by an indirect, allosteric mechanism (Moazed and Noller, 1986; Stern et al., 1988c). Its association with A-site function is reinforced by the discovery of ochre suppression by a mutant of yeast mitochondria containing a G to A mutation at a position corresponding to G5 17 of E. coli 16S rRNA (Shen and Fox, Oxford University Press
1989), by the occurrence of streptomycin-resistance mutations in the 530 loop (Gauthier et al., 1988; Fromm et al., 1989), and by enhancement of reactivity of C525 toward dimethyl sulfate by aminoglycoside antibiotics (Moazed and Noller, 1987). Recently, we have demonstrated that point mutations involving G530 confer a dominant lethal phenotype, and that ribosomes containing such mutations are impaired in some translational step following initiation, consistent with the involvement of this strucure in A-site function (Powers and Noller, 1990). Precisely how the 530 loop might act is not at all understood, however, and is a particularly intriguing question in view of its apparent remoteness from the decoding site. Recently, Woese and Gutell (1989) have proposed an unusual higher-order structural interaction involving the 530 loop, based on comparative sequence analysis. This interaction involves base pairing between residues 524-526 in the 530 region hairpin loop with residues 505-507 in the adjoining 510 region bulge loop (Figure 1). The resulting structure is an example of the class of RNA strucures called pseudoknots (Pleij et al., 1985). These structures appear to be rare in rRNA (Gutell and Woese, 1990), and so the occurrence of a pseudoknot in such a functionally interesting context in 16S rRNA raises the possibility that it may play a functional role. Because of the extremely high conservation of primary structure in the 530 loop, sequence variation supporting the proposed structure is sparse. Furthermore, comparative analysis, although a powerful tool for analysis of RNA structure, does not inform us about the specific nature of the biological requirement for a structure. In this paper, we demonstrate directly, by site-directed mutagenesis, the requirement of this interaction for the proper functioning of the ribosome. In addition, we find that certain perturbations of the pseudoknot structure confer resistance to streptomycin, a drug that is known to interfere with the decoding process.
Results Genetic analysis of the proposed 530 stem -loop tertiary interaction The interaction involving nucleotides 505-507 and 524-526 in 16S rRNA, proposed by Woese and Gutell (1989), is shown in Figure 1.We took a genetic approach to test the existence of this interaction by reasoning that if it is essential for ribosome function, then mutations that disrupt pairing between these bases should impair cell growth, while compensatory changes that restore pairing should restore wild-type-like growth. We constructed mutations based on the sequences shown in Figure 1. As each of these variations is known to occur in two or more different eubacterial and/or mitochondrial 16S-like rRNAs, we believed they had the best potential for functioning in E. coli. Site-directed mutations were introduced into plasmid 2203
T.Powers and H.F.Noller AGCCG G 5C G-
A
GC
520-A
U A
C
CG * UA U-A
510 C-G \ C-G AAU -A U
C-G-540
GC G CGGC-
524-526 507-505
232
35
GUC CAG
3
0
ACC UGG
0
0
GCC CGG
43
GCA CGU
G
A-U C-G G-C
500
Fig. 1. (Left) Secondary structure of the 530 stem-loop region in 16S rRNA from E.coli. The tertiary interaction between positions 505-507 and 524-526, proposed by Woese and Gutell (1989), is shown in bold. (Right). Sequence variations found in two or more 16S or 16S-like rRNAs that support the proposed interaction. Differences from the E.coli sequence are shown in bold. Numbers under each phylogenetic group indicate the number of known variants. Data from Woese and Gutell (1989) and R.R.Gutell (personal communication).
pSTL102 (Triman et al., 1989), as described in Materials and methods. This plasmid contains an ampicillin-resistance gene and the entire rrnB operon from E. coli. A C to U transition at position 1192 in the 16S rRNA gene confers resistance to spectinomycin, allowing for the phenotypic characterization of plasmid-encoded 16S rRNA. E. coli strain DH1 was transformed with the mutant plasmids and the growth of these resulting strains was compared in the presence of spectinomycin (Spc) and/or ampicillin (Amp). As controls, cells were also transformed with plasmids pSTL102 and pKK3535, the latter being the Spcs parent to pSTL102. For each mutant, the amount of 16S rRNA assembled into ribosomes and polyribosomes was determined, using the U1 192 mutation to identify plasmidencoded RNA in a quantitative primer extension assay (Sigmund et al., 1988). Below we describe our results for each set of mutations examined. G506 - C525 interaction The most convincing sequence variation suggesting an interaction between the 510 and 530 regions involves pairing between G506 and C525. The sequence A506 - U525 is found within the eubacteria, in the gram-positive mycoplasmas, as well as in several different mitochondrial 16S-like rRNAs (Woese and Gutell, 1989; Figure 1). Cells carrying the U525 single mutation grew normally on Amp plates but displayed a slight reduction in colony size on Spc plates, at higher temperatures (Table I and Figure 2, pU525). Plasmid-encoded 16S rRNA from these cells was present at wild-type levels in both ribosomes and polyribosomes (data not shown). No transformants were recovered with the plasmid containing the A506 single mutation, suggesting that expression of this mutation was lethal to the host cell. We tested this directly by placing this mutation under inducible expression, utilizing the PL promoter/operator in plasmid pLK45 and the temperature-sensitive cI repressor from bactriophage lambda (Powers and Noller, 1990) (see Materials and methods). The results of induction experiments are shown in Table II. Cells transformed with plasmid pLA506 displayed a slow-growth phenotype at 420C, the induced temperature. Analysis of the distribution of plasmidencoded rRNA, using the U1 192 marker as above, showed a substantially reduced amount of mutant 16S rRNA in both ribosomes and polyribosomes (Table II). We have determined previously that the amount of plasmid rRNA
2204
incorporated into ribosomes from the PL promoter in pLK45 is substantially less (50% versus >70%, respectively) than from the natural PIP2 promoters in pSTL102 (Powers and Noller, 1990). We believe this difference in gene-dosage explains why pLK45 does not confer resistance to spectinomycin (our unpublished results). This difference may also explain why the A506 mutation is apparently lethal in pSTL102 but viable, albeit slowgrowing, under PL control. In contrast, when the A506 and U525 mutations were combined, cells grew similarly to wild-type on both Amp and Amp+Spc plates (Table I and Figure 2, pA506/U525). Plasmid-encoded 16S rRNA was also present at wild-type levels in both ribosomes and polyribosomes (data not shown). These results demonstrate that the U525 mutation can suppress the severe growth-defect caused by the A506 mutation and that the A506 mutation can suppress the weak growth-defect caused by the U525 mutation. Recently, a C to U mutation at position 525 (E. coli numbering) in tobacco chloroplast 16S rRNA has been shown to confer resistance to streptomycin (Sm) (Fromm et al., 1989). However, cells carrying pU525 failed to grow on Amp plates containing 3 mg/l Sm, the minimum concentration of Sm required to prevent the growth of cells containing wild-type plasmids (Figure 2). This result is consistent with the difficulty other groups have experienced in expressing 16S rRNA Smr mutations in E. coli (Montandon et al., 1986; Melanqon et al., 1988). This is most likely due to the fact that sensitivity to streptomycin is dominant in cells heterozygous for Smr and Sm5 alleles (Lederberg, 1951). It has been observed, however, that by combining the Ul 192 Spcr marker and a putative Smr mutation in cis, cells grow in the presence of both spectinomycin and streptomycin; spectinomycin presumably inactivates the sensitive, chromosomally-encoded ribosomes, allowing expression of the recessive streptomycin resistance (E.Morgan, personal communication). Accordingly, significant growth was observed on Amp + Spc + Sm plates by cells carrying U525, but not by cells carrying the control plasmids nor the A506/U525 double mutation (Figure 2 and Table I). Cells appearing on these plates were re-tested on selective plates and were found to maintain their original phenotype. Most importantly, no growth was observed on plates containing Sm alone, indicating that these cells had not acquired a chromosomal mutation conferring streptomycin resistance. These results indicate that resistance to
A functional pseudoknot in 16S ribosomal RNA Table I. Growth properties of 530 region pseudoknot mutants on selective platesa
Plasmid
510/530 interaction
Temperature
26°C Amp
AmpSpc
370C AmpSpcSm Amp
AmpSpc
+++
+++
-
+++
+++
+++
+++
++
+++
++
+++
+++
_
+++
+
+l-
_
+1I-
-
+++
420C Amp
AmpSpc
AmpSpcSm
+++
+++
-
++
+++
++
++
+++
-
+++
++
_
+
+
_
+
+
+
_
+1I-
-
_
+1I-
-
_
++
-
+++
++
-
+++
