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Journal of Bacteriology, September 2002, p. 5052-5057, Vol. 184, No. 18
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.18.5052-5057.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Mutational Evidence for a Functional Connection between Two Domains of 23S rRNA in Translation Termination

Alexey L. Arkov,^, Klas O. F. Hedenstierna, and Emanuel J. Murgola^*

Department of Molecular Genetics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030

Received 20 December 2001/ Accepted 21 June 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
Nucleotide 1093 in domain II of Escherichia coli 23S rRNA is part of a highly conserved structure historically referred to as the GTPase center. The mutation G1093A was previously shown to cause readthrough of nonsense codons and high temperature-conditional lethality. Defects in translation termination caused by this mutation have also been demonstrated in vitro. To identify sites in 23S rRNA that may be functionally associated with the G1093 region during termination, we selected for secondary mutations in 23S rRNA that would compensate for the temperature-conditional lethality caused by G1093A. Here we report the isolation and characterization of such a secondary mutation. The mutation is a deletion of two consecutive nucleotides from helix 73 in domain V, close to the peptidyltransferase center. The deletion results in a shortening of the CGCG sequence between positions 2045 and 2048 by two nucleotides to CG. In addition to restoring viability in the presence of G1093A, this deletion dramatically decreased readthrough of UGA nonsense mutations caused by G1093A. An analysis of the amount of mutant rRNA in polysomes revealed that this decrease cannot be explained by an inability of G1093A-containing rRNA to be incorporated into polysomes. Furthermore, the deletion was found to cause UGA readthrough on its own, thereby implicating helix 73 in termination for the first time. These results also indicate the existence of a functional connection between the G1093 region and helix 73 during translation termination.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The rRNAs from both subunits have been implicated in various stages of translation (3, 22, 24, 25, 36). The importance of rRNA for the last step of translation, peptide chain termination, has been demonstrated by in vivo and in vitro experiments (1, 4, 8, 10, 17, 20, 27, 34). In particular, the G1093A mutation in domain II of Escherichia coli 23S rRNA (Fig. 1, top), isolated as a codon-specific suppressor of UGA mutations, was shown to cause defects in the catalysis of peptidyl-tRNA hydrolysis in the presence of release factor 2 (RF2) in a realistic in vitro translation system (1). (RF2 mediates termination at UGA and UAA, while RF1 functions at UAG and UAA [18, 29].) In addition to the catalytic defect caused by G1093A, this mutation also affects binding of RF2 to the ribosome (4), clearly suggesting that defective termination contributes to the readthrough of nonsense codons detected in vivo. Nucleotide G1093 is in the highly conserved structure historically referred to as the GTPase center (also the binding region for large-subunit ribosomal protein L11). In this study, we asked if there are sites elsewhere in 23S rRNA that are functionally associated with G1093 during translation termination. These sites could interact either directly or indirectly with G1093. An indirect interaction could, for example, be transmitted by RF2. To identify such sites, we took advantage of the fact that G1093A, when expressed from a multicopy plasmid, is lethal at temperatures above 37°C (21). After DNA segment-targeted PCR random mutagenesis, we selected for 23S rRNA mutants that would reverse the high-temperature sensitivity. The present study describes the isolation and characterization of a 2-nucleotide (nt) deletion in helix 73 that reverses the mutant phenotypes of G1093A. The results demonstrate the existence of a functional connection between the G1093 region and helix 73 during translation termination.

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FIG. 1. Locations of mutations described in this study. (Top) Outline of 23S rRNA secondary structure (16) with helix 73 shown by thick black lines. The G1093A mutation is indicated. (Bottom) Detail of helix 73 showing the location of the 2-nt deletion that reverses the high-temperature lethality of the G1093A mutation.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Plasmids used in this study. Plasmid pNO2680 was derived from pBR322 and contains the wild-type rRNA operon rrnB under the control of the bacteriophage lambda P_L promoter (15). Plasmid pNO1093 was derived from pNO2680 by introducing the G1093A mutation into the 23S rRNA gene. A low-expression plasmid, pL1093, was derived from pNO1093 by introducing mutations that weaken the P_L promoter (13, 19, 23). The promoter mutations, introduced by site-directed mutagenesis and verified by sequencing, were A to G at position -9 (13), C to T at position -31 (also known as sex1) (19, 23), and a change of the AATT tetranucleotide at positions -52 to -49 to GGCC (13). The decreased expression of the A1093-containing rRNA from the altered P_L promoter was confirmed by primer extension analysis essentially as described previously (32). For a control, the same promoter mutations were introduced in pNO2680, resulting in plasmid pL2680. Plasmid pNO17sm is identical to pNO2680 except for the second-site mutation in helix 73 of the 23S rRNA gene isolated in this study (see Results). Plasmid pNO17dm contains both the G1093A mutation and the second-site mutation.

Selection for second-site mutations that compensate for high temperature sensitivity caused by G1093A. To obtain second-site mutations that compensate for temperature-conditional lethality caused by G1093A, we introduced random mutations into the I-CeuI-Bpu1102I segment of the rrnB operon in pNO1093 by subjecting it to PCR mutagenesis. This segment comprises the 3' part of domain IV as well as domains V and VI of 23S rRNA, 5S rRNA, and the transcription termination region. The PCR was performed under standard conditions as described previously (38). Oligomers PS9 (5'-AGGTGGTCAGGTAGAGAATA-3'), annealing upstream of the I-CeuI site, and PS10 (5'-CGCATCAGGTTCATCATGAA-3'), annealing downstream of the Bpu1102I site, were used as PCR primers. After the mutagenized I-CeuI-Bpu1102I fragments were inserted into pNO1093, survivors were selected at 41°C in E. coli strain AL1 [glyV55 recA/F' trpA(UGA115)] (2). Glucose minimal (GM) medium supplemented with 10 µg of indole per ml (indole supports growth of mutant trpA strains) and 100 µg of ampicillin per ml was used to select for the mutations.

Analysis of the selected mutants. Plasmids isolated from survivors were digested with a series of restriction endonucleases to verify that the plasmids did not contain any large-scale rearrangements. The I-CeuI-Bpu1102I segments from these plasmids were sequenced and recloned in pNO1093, and the ability of the recloned segments to confer survival at 41°C in the presence of G1093A was verified. To evaluate the properties of the selected compensatory mutations alone, the I-CeuI-Bpu1102I segments with the second-site mutation were subcloned into pNO2680 in place of the wild-type I-CeuI-Bpu1102I segment. Cloning procedures were performed essentially as described previously (31). DNA sequence analysis was performed at the automated DNA sequencing core facility of the Department of Microbiology and Molecular Genetics of The University of Texas Medical School at Houston and at the M. D. Anderson Cancer Center's automated sequencing facility.

In vivo tests. The rRNA mutations were tested in vivo for their ability to suppress various mutant alleles of trpA, which encodes the subunit of tryptophan synthetase. In these test strains, the chromosomal copy of the trp operon has been deleted (21), and the different trpA genes are instead carried on the Fredericq episome, which contains the cysB trp tonB region of the chromosome (12). Readthrough of nonsense codons was tested for all three nonsense codons (UAA, UGA, and UAG) at each of four codon positions (positions 15, 115, 211, and 243) in trpA. Similarly, the ability of the rRNA mutations to cause misreading of several sense codons was tested by monitoring for missense suppression of four mutant codons, GAA, AGA, AAG, and UGG, at position 211. We also determined whether the rRNA mutations were able to cause ribosomal frameshifting by testing three frameshift mutations, the +1 mutations trpA8 (33) and trpE9777 (7) and the -1 mutation trpE91 (5). The trpA8 and trpE9777 alleles are chromosomal. Suppression was usually monitored on GM medium alone or on GM medium supplemented with a low concentration of tryptophan (40 ng/ml). As this amount of added tryptophan is insufficient to sustain growth by itself, growth on these media indicates that the rRNA mutations are able to suppress the trpA mutations (2). Supplementing the GM medium with tryptophan proved to increase the sensitivity of detecting the Trp⁺ phenotype, presumably because it reduced the amount of tryptophan that the cells were required to synthesize (2). The medium used for monitoring general growth was usually GM medium supplemented with a high concentration of tryptophan, 20 µg/ml. For mutants containing the helix 73 deletion mutation with or without G1093A, the tests were performed at 28, 35, 37, and 42°C. By itself, the G1093A mutant was monitored at 28 and 35°C, which are permissive temperatures for this mutant. Specific growth conditions different from those described in this section are described at the first mention in Results. The growth of a given strain was estimated from the growth of a patch of that strain on agar plates essentially as described previously (2, 9, 33).

Determination of relative amounts of mutant rRNA in ribosome fractions. Large subunits (50S) and translating ribosomes (polysomes) were isolated from strain AL1 carrying plasmids expressing the various 23S rRNA mutations. Bacteria were lysed as described previously (14) while growing exponentially at either 31 or 42°C in Luria-Bertani (LB) medium (9). Ribosomal fractions were separated by centrifuging the lysate through a 10 to 40% (wt/vol) linear sucrose density gradient (1). rRNA from various fractions was extracted sequentially with phenol, phenol-chloroform, and chloroform and then precipitated with ethanol.

The amount (percent) of mutant rRNA isolated from subunits or polysomes of a given mutant was determined by primer extension analysis essentially as described previously (32). Autoradiograms were scanned by an ARCUS II scanner (AGFA) and analyzed with software from Bio Image (Intelligent Quantifier, version 2.2.1). Alternatively, phosphorimaging was used to analyze primer extension data (PhosphorImager S1; Molecular Dynamics).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of a second-site mutation. The selection for secondary mutations in 23S rRNA able to compensate for the temperature-conditional lethality caused by G1093A yielded several isolates. A number of these isolates contained large deletions, presumably preventing any incorporation of the rRNA into ribosomes, and some contained multiple mutations. However, one isolate was found to contain a single mutation. This mutation, consisting of a deletion of 2 nt in helix 73 shortening the sequence CGCG between positions 2045 and 2048 to CG (Fig. 1), was selected for further characterization. This deletion by itself was viable at high temperatures (Fig. 2).

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FIG. 2. Growth phenotype and UGA readthrough conferred by the rRNA mutants. E. coli strain AL1, which contains a trpA gene with a stop codon (UGA) at position 115 (Materials and Methods), was transformed with the mutant plasmids. Patches are numbered according to the mutations (plasmids shown in the parentheses) as follows: 1, G1093A (pNO1093); 2, wild type (pNO2680); 3, wild type, low-expression promoter (pL2680); 4, G1093A, low-expression promoter (pL1093); 5, G1093A and helix 73 deletion (pNO17dm); and 6, helix 73 deletion (pNO17sm). Readthrough plates contain GM medium enriched with 5 mg of acid casein hydrolysate (ACH) per ml. Growth plates contain GM medium enriched with 5 mg of ACH per ml and 20 µg of tryptophan per ml. (ACH contains all the common amino acids except tryptophan.) All plates also contain 100 µg of ampicillin per ml. Patches were grown on GM medium supplemented with Trp (20 µg/ml) and replicated to the readthrough and growth plates, which were subsequently incubated at 31 and 42°C. Growth plates were photographed after 2 days of incubation. Readthrough plates were photographed after 3 days of incubation.

Effects of the helix 73 deletion on UGA readthrough caused by G1093A. The G1093A mutation (expressed from the P_L promoter in pNO1093) confers a high level of readthrough of UGA115 in the trpA gene at 31°C (this temperature is permissive for growth) (Fig. 2). Under the same conditions, neither the double mutant containing the helix 73 deletion and G1093A nor the helix 73 deletion by itself showed any UGA readthrough (Fig. 2). When the expression level was lowered by introducing several point mutations in the P_L promoter (Materials and Methods), the G1093A mutant became viable at 42°C and showed efficient readthrough of UGA115 at both 31 and 42°C (Fig. 2).

Measurement of the amount of mutant rRNAs in ribosomal fractions. A potential explanation for how the deletion in helix 73 could eliminate the high temperature sensitivity and dramatically lower the UGA readthrough caused by G1093A could be that the deletion reduces the amount of mutant rRNA in active ribosomes (polysomes). The polysome incorporation of rRNA containing both G1093A and the helix 73 deletion was approximately 8% at 31°C, which is about four times lower than that of rRNA containing G1093A alone (Table 1). However, lowering the amount of G1093A-containing polysomes to about 2% by introducing point mutations in the P_L promoter (Table 1) still produced a high level of readthrough at both 31 and 42°C (Fig. 2). The decrease in incorporation to 8% is therefore clearly not sufficient to explain the failure to observe UGA readthrough with the double mutant under the same conditions. The substantial amount (20%) of doubly mutant rRNA present in polysomes at 42°C also supports the conclusion that the reversal of phenotypes brought about by the helix 73 deletion cannot be explained entirely by lowered incorporation (Table 1).

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TABLE 1. Amount of mutant rRNA in large subunits and polysomesa

The amount of mutant rRNA in polysomes isolated from bacteria expressing the helix 73 deletion alone was similar to that found in polysomes isolated from the double mutant with both the deletion and G1093A (Table 1). Furthermore, in 50S subunits, the amount of rRNA containing the helix 73 deletion, either alone or together with G1093A, was about two times higher than that of G1093A-containing rRNA (Table 1). That increase was consistent with the fact that the amount of 50S subunits isolated from either of these two mutants was reproducibly larger than that from either the G1093A mutant or the wild type (data not shown).

UGA readthrough caused by the helix 73 deletion. Our finding that the helix 73 deletion decreased the high level of UGA readthrough caused by G1093A to undetectable levels (Fig. 2) without preventing rRNA from being incorporated into the polysomes (Table 1) indicated a functional connection between the G1093 region and helix 73 (Fig. 1). This suggested that the helix 73 deletion alone might affect the same ribosomal function and cause readthrough of stop codons. As discussed above, the helix 73 deletion by itself did not confer any detectable readthrough of UGA on GM medium. However, when GM medium supplemented with a low concentration of tryptophan was used, the helix 73 deletion by itself caused readthrough of UGA115 (Fig. 3) and UGA15 (data not shown). All three stop codons were tested at each of the four codon positions (positions 15, 115, 211, and 243). The helix 73 deletion did not cause readthrough of any other stop codon. (The rationale behind the addition of a low concentration of tryptophan to increase the sensitivity is explained in Materials and Methods.) Furthermore, compared with the recA mutant strain AL1, readthrough of UGA115 was more pronounced in the more robust recA⁺ strain shown in Fig. 3. The deletion caused neither misreading nor frameshifting. Like the deletion alone, the double mutant containing the helix 73 deletion together with G1093A showed readthrough of UGA115 (Fig. 3) and UGA15 (data not shown) on the supplemented medium, but no readthrough of other nonsense codons, misreading of sense codons, or frameshifting (data not shown).

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FIG. 3. UGA readthrough caused by the helix 73 deletion. E. coli strain FTP5657 [glyV55/F' trpA(UGA115)], which is a recA+ analog of strain AL1, was transformed with plasmids expressing the rRNA mutations. Patches are numbered according to the mutations (plasmids shown in the parentheses) as follows: 1, G1093A, low expression (pL1093); 2, wild type, low expression (pL2680); 3, G1093A and helix 73 deletion (pNO17dm); and 4, helix 73 deletion (pNO17sm). Growth plates contain GM medium supplemented with 20 µg of tryptophan per ml. Readthrough plates contain GM medium supplemented with 40 ng of tryptophan per ml. All plates also contain 100 µg of ampicillin per ml. Plates were incubated at 31°C and photographed after 2 days.

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study describes the isolation and characterization of a deletion in helix 73 of E. coli 23S rRNA that suppresses the temperature-conditional lethality and UGA readthrough caused by the G1093A mutation in domain II. In addition, the deletion by itself was shown to cause UGA readthrough. Helix 73 is located approximately in the center of the large subunit (6, 36), some 75 Å from the G1093 region. Thus, a direct interaction between the two regions cannot be invoked to explain the suppression of the temperature-conditional lethality. One end of helix 73, defined by the 2057/2611 base pair, is close to the peptidyltransferase center (the binding site for the puromycin moiety of CCdAp-puromycin is approximately 20 Å from this base pair). This end of the helix is also exposed to the exit channel (24), and mutations in the 2057/2611 base pair and adjacent nucleotides confer resistance to antibiotics that inhibit peptidyltransferase (30). The other end of helix 73 is directed towards the side of the subunit containing the G1093 region (the GTPase center or L11-binding region).

Since termination at UGA is mediated by RF2 and UAA and UAG can be decoded by RF1, the UGA-specific readthrough caused by the deletion suggests a defect in RF2-dependent termination. The G1093A mutation was also shown to cause UGA-specific readthrough under certain conditions in vivo (17) and to affect mainly RF2-dependent termination in vitro (1, 4). Therefore, these results strongly implicate helix 73 in RF2-dependent termination. A connection between helix 73 and RF2-dependent termination is in fact consistent with previous experiments that showed that the expression of an antisense rRNA fragment complementary to nt 735 to 766 of 23S rRNA induces UGA-specific readthrough in vivo (11). The region covered by the fragment essentially corresponds to helix 35 (nt 736 to 760), and the recent high-resolution structures of the large subunit (6, 36) show that the loop capping this helix is in close proximity to helix 73. Specifically, the bases of U2613 in helix 73 and U747 (m⁵U) in the loop capping helix 35 are stacked, and these 2 nt also interact with 2 neighboring nucleotides in the central loop (A2013 and A2014, respectively), each interaction involving two hydrogen bonds (6). As the 2-nt deletion would significantly alter the structure of helix 73, its interaction with helix 35 would most likely be disturbed. It is therefore possible that the cause of the UGA readthrough in both these cases is the disruption of the interaction between helices 35 and 73.

Other long-range intramolecular contacts involving helix 73 include its interaction with the loop capping helix 100 (nt 2815 to 2831) (6). nt 2823 to 2826 in this loop interact through hydrogen bonds with nt 2047, 2622, and 2623 in helix 73. Since nt 2047 is within the CGCG tetranucleotide where the 2-nt deletion is located and nt 2622 and 2623 are on the opposite strand, this interaction is most likely abolished. However, to our knowledge, no specific function has been associated with helix 100, so the relevance, if any, of this interaction for RF2-dependent termination is unclear. Finally, extensive sections of proteins L3 and L22 are in close proximity to the minor groove of helix 73. Mutations in Saccharomyces cerevisiae ribosomal protein L3 have been reported to affect frameshifting (28), but we are not aware of any reports linking either of these proteins to termination.

The catalytic site for peptidyl-tRNA hydrolysis must be located close to, or could possibly be identical to, the peptidyltransferase center. The location of helix 73 near the peptidyltransferase center is thus consistent with a role in triggering peptidyl-tRNA hydrolysis. However, our results also indicate that there is a specific functional connection between the G1093 region and helix 73 during RF2-mediated translation termination. This is indicated by the fact that the helix 73 deletion, which by itself is impaired in UGA termination, nonetheless is able to reverse the UGA readthrough displayed by the G1093A mutation. If the two regions operated independently at different steps in the termination pathway, one would have expected that combining G1093A and the helix 73 deletion would enhance UGA readthrough compared with either mutation on its own. As the locations of the two regions in the crystal structures (6, 36) preclude a direct interaction, the functional interdependence must be mediated through other parts of the ribosome, through RF2, or both.

A connection between helix 73 and the GTPase-associated region could potentially be established through helix 89 (nt 2455 to 2496) whose tip is close to and possibly in contact with the GTPase-associated region (36). The potential involvement of helix 89 is also supported by the observation that a nucleotide deletion in helix 89 reverses the UGA readthrough (but not the temperature-conditional lethality) associated with A1093 (37). This deletion thus resembles the 2-nt deletion in helix 73 in its ability to reverse the phenotype of A1093. In addition, the potential involvement of helix 89 in termination is also suggested by the fact that several mutations in this helix cause readthrough of stop codons (26, 37) although not with the same specificity as the helix 73 deletion.

Alternatively, since G1093 is an "RF2-interactive site" (35), involved in RF2 binding (4), the G1093A mutation might affect the positioning of RF2 on the ribosome. This in turn could influence the ability of RF2 to participate in the transmission of the termination signal to the hydrolytic center of the large subunit, perhaps through the mediacy of helix 73.

 
We thank Amos Oppenheim and Gary Gussin for advice on constructing mutations that decrease the efficiency of the P_L promoter and all members of the Murgola laboratory for discussions. We also thank Walter J. Pagel for helpful editorial comments.

This work was supported in part 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 in part by grant CA16672 from the National Cancer Institute.

 
* Corresponding author. Mailing address: Molecular Genetics (Box 11), M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Phone: (713) 792-8939. Fax: (713) 794-4295. E-mail: mannyj{at}mdanderson.org.

Present address: Howard Hughes Medical Institute/Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY 10016.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, September 2002, p. 5052-5057, Vol. 184, No. 18
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.18.5052-5057.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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