Proceedings of the National Academy of Sciences of the United States of America, Jun 1, 1988
We have isolated an unusual codon-specific translational suppressor in Escherichia coli. The supp... more We have isolated an unusual codon-specific translational suppressor in Escherichia coli. The suppressor resulted from a spontaneous mutation in a chromosomal gene during a selection for suppressors of the auxotrophic nonsense mutation trpA(UGA211). The suppressor allows readthrough of UGA mutations at two positions in trpA and at two sites in bacteriophage T4. It does not, however, suppress amber (UAG) or ochre (UAA) mutations that were tested in both genomes, some of which were at the same positions as the suppressible UGA mutations. The suppressor also does not allow mistranslation of the UGA-related tqpA missense mutations UGG at
Proceedings of the National Academy of Sciences, 1991
Site-directed mutagenesis was performed on a sequence motif within the 3' major domain of Esc... more Site-directed mutagenesis was performed on a sequence motif within the 3' major domain of Escherichia coli 16S rRNA shown previously to be important for peptide chain termination. Analysis of stop codon suppression by the various mutants showed an exclusive response to UGA stop signals, which was correlated directly with the continuity of one or the other of two tandem complementary UCA sequences (bases 1199-1204). Since no other structural features of the mutated ribosomes were hampered and the translation initiation and elongation events functioned properly, we propose that a direct interaction occurs between the UGA stop codon on the mRNA and the 16S rRNA UCA motif as one of the initial events of UGA-dependent peptide chain termination. These results provide evidence that base pairing between rRNA and mRNA plays a direct role in termination, as it has already been shown to do for initiation and elongation.
Proceedings of the National Academy of Sciences, 1983
In a previous publication, an unusual UGG-reading missense suppressor caused by insertion of an e... more In a previous publication, an unusual UGG-reading missense suppressor caused by insertion of an extra adenylate residue in the anticodon loop of an Escherichia coli glycine tRNA was described. In this study, we provide in vivo evidence that the additional nucleotide causes an "anticodon shift" by one nucleotide in the 3' direction and that the "new" anticodon can explain the unanticipated coding properties of the suppressor. We converted the UGG suppressor with ethyl methanesulfonate, a base-substitution mutagen, to suppressors that read codons related to UGG by a single base change. Sequence analysis of each mutant tRNA revealed that its mutational alteration was an anticipated base change in one of the three nucleotides of the "new" anticodon. Although the new suppressors read codons beginning with A or U, the mutant tRNAs lack the customary hypermodified nucleosides on the 3' side of the anticodon. As determined on the basis of their in vivo ...
Beginning with a missense suppressor tRNA and a nonsense suppressor tRNA, both in Escherichia col... more Beginning with a missense suppressor tRNA and a nonsense suppressor tRNA, both in Escherichia coli and each containing an extra nucleotide in the anticodon loop, we generated new suppressors in vivo by spontaneous deletion of specific nucleotides from the anticodon loop. In one experiment, the new suppressor was generated by a double mutational event, base substitution and nucleotide deletion. A novel ochre suppressor is also described. It is very efficient in nonsense suppression but has no ms2i6 modification of the A residue on the 3' side of the anticodon. The results have important implications for tRNA structure-function relationships, tRNA recognition by tRNA-modifying enzymes, mechanisms of deletion mutation, and tRNA evolution.
Previous studies have demonstrated many amino acid and codon substitutions at position 211 of the... more Previous studies have demonstrated many amino acid and codon substitutions at position 211 of the alpha chain of tryptophan synthetase of Escherichia coli. In order to extend our studies on suppressor tRNAs and, in general, on accuracy in the translation of genetic information, we have devised specific selections for sense and nonsense codons corresponding to alpha chain position 234. Using codon-specific suppressors and selection for missense revertants of nonsense mutations, we have brought to 18 (from six) the number of codons at 234. This number includes all three nonsense codons and ten (of the remaining 15) that code for nonfunctional amino acids at that position. Furthermore, by adding to these results the testing of the suppressibility of trpA(234) nonsense mutations by suppressor tRNAs of known aminoacylation specificity, we have increased to 12 (from five) the number of amino acids whose functionality at position 234 is known. The existence of trpA mutants with so many amino acid substitutions at two positions in the tryptophan synthetase alpha chain should prove helpful for structure-function studies of that protein. Furthermore, the availability, at both positions, of at least 12 codons (including the three nonsense codons) that result in a nonfunctional alpha chain allows the selection of mutations that affect the suppression of a given codon as it occurs in two different mRNA contexts. Studies with such mutants should help elucidate the role in translational accuracy of mRNA secondary structure, tRNA-tRNA interactions on the ribosome, and interactions of the ribosome with tRNA, mRNA, and, in the case of nonsense suppression, peptidyl release factors.
RNA sites that contain unpaired or mismatched nucleotides can be interaction sites for other macr... more RNA sites that contain unpaired or mismatched nucleotides can be interaction sites for other macromolecules. C1054, a virtually universally conserved nucleotide in the 16 S (small subunit) ribosomal RNA of Escherichia coli, is part of a highly conserved bulge in helix 34, which has been located at the decoding site of the ribosome. This helix has been implicated in several translational events, including peptide chain termination and decoding accuracy. Here, we observed interesting differences in phenotype associated with the three base substitutions at, and the deletion of, nucleotide C1054. The phenotypes examined include suppression of nonsense codons on different media and at different temperatures, lethality conditioned by temperature and level of expression of the mutant rRNA, ribosome pro®les upon centrifugation through sucrose density gradients, association of mutant 30 S subunits with 50 S subunits, and effects on the action of tRNA suppressor mutants. Some of our ®ndings contradict previously reported properties of individual mutants. Particularly notable is our ®nding that the ®rst reported 16 S rRNA suppressor of UGA mutations was not a C1054 deletion but rather the base substitution C1054A. After constructing ÁC1054 by site-directed mutagenesis, we observed, among other differences, that it does not suppress any of the trpA mutations previously reported to be suppressed by the original UGA suppressor. In general, our results are consistent with the suggestion that the termination codon readthrough effects of mutations at nucleotide 1054 are the result of defects in peptide chain termination rather than of decreases in general translational accuracy. The phenotypic heterogeneity associated with different mutations at this one nucleotide position may be related to the mechanisms of involvement of this nucleotide, the two-nucleotide bulge, and/or helix 34 in particular translational events. In particular, previous indications from other laboratories of conformational changes associated with this region are consistent with differential effects of 1054 mutations on RNA-RNA or RNA-protein interactions. Finally, the association of a variety of phenotypes with different changes at the same nucleotide may eventually shed light on speculations about the coevolution of parts of ribosomal RNA with other translational macromolecules.
After our first observation of codon context effects in missense suppression ( Murgola & ... more After our first observation of codon context effects in missense suppression ( Murgola & Pagel , 1983), we measured the suppression of missense mutations at two positions in trpA in Escherichia coli. The suppressible codons in the trpA messenger RNA were the lysine codons, AAA and AAG, and the glutamic acid codons, GAA and GAG. The mRNA sites of the codons correspond to amino acids 211 and 234 of the trpA polypeptide, positions at which glycine is the wild-type amino acid. Our data demonstrated codon context effects with both pairs of codons. The results indicate that suppression of AAA and AAG by mutant lysine transfer RNAs was more efficient at 211 than at 234, whereas suppression of GAA and GAG by two different mutant glycine tRNAs was more efficient at 234 than at 211. In general, the context effects were more pronounced with GAG and AAG than with GAA and AAA. (In some instances it appeared that suppression of GAA or AAA at a given position was more effective than suppression of GAG or AAG.) By contrast, no context effects were observed with a glyT suppressor of AAA and AAG, a glyT GAA/G-suppressor, and a glyU suppressor of GAG. Our observation of this phenomenon in missense suppression demonstrates that codon context can affect polypeptide elongation and that the effects can be different depending on the codons and tRNAs examined. It is suggested that tRNA-tRNA interaction on the ribosome is involved in the observed context effects.
A modified assay has been devised for the physiological reaction, indole-3-glycerol phosphate to ... more A modified assay has been devised for the physiological reaction, indole-3-glycerol phosphate to Trp, of the enzyme tryptophan synthetase. The assay may be applied to crude bacterial extracts, and is based on the measurement of incorporation of radioactivity from [3H]Ser into Trp. Comparison with previous colorimetric assays indicates an improvement in sensitivity of about 30-fold, and advantages in terms of sample economy and simplified manipulation.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1990
The base sequence around nonsense codons affects the efficiency of nonsense codon suppression. Pu... more The base sequence around nonsense codons affects the efficiency of nonsense codon suppression. Published data, comparing different nonsense sites in a mRNA, implicate the two bases downstream of the nonsense codon as major determinants of suppression efficiency. However, the results we report here indicate that the nature of the contiguous upstream codon can also affect nonsense suppression, as can the third (wobble) base of the contiguous downstream codon. These conclusions are drawn from experiments in which the two Set codons UCU233 and UCG235 in a nonsense mutant form (UGA234) of the trpA gene in Escherichia coil have been replaced with other Ser codons by site-directed mutagenesis. Suppression of these trpA mutants has been studied in the presence of a UGA nonsense suppressor derived from glyT. We speculate that the non-site-specific effects of the two adjacent downstream bases may be largely at the level of the termination process, whereas more site-specific or codon-specific effects may operate primarily on the activity of the suppressor tRNA.
To screen for ribosomal RNA mutants defective in peptide chain termination, we have been looking ... more To screen for ribosomal RNA mutants defective in peptide chain termination, we have been looking for rRNA mutants that exhibit different patterns of suppression of nonsense mutations and that do not suppress missense mutations at the same positions in the same reporter gene. The rRNA mutations were induced by segment-directed randomly mutagenic PCR treatment of a cloned rrnB operon, followed by subcloning of the mutagenesis products and transformation of strains containing different nonsense mutations in the Escherichia coli trpA gene. To date, we have repeatedly obtained only two small sets of mutations, one in the 3′ domain of 16S rRNA, at five nucleotides out of the 610 mutagenized (two in helix 34 and three in helix 44), and the other in 23S rRNA at only four neighboring nucleotide positions (in a highly conserved hexanucleotide loop) within me 1.4 kb mutagenized segment. There is variety, however, in the suppression patterns of the mutants, ranging from suppression of UAG or UG...
m This paper describes a novel mechanism for reversion of nonsense mutations in the trpA gene of ... more m This paper describes a novel mechanism for reversion of nonsense mutations in the trpA gene of F~cherichia coli. This mechanism, deletion of the nonsense codon, was discovered in the course of select _~ for missense revertants of trpA(UGA211) and for catalytically active tryptophan synthetase alpha chain revertants of trpA(UAA234) and trpA(UAG234). Each type of revertant trpA was cloned and its DNA sequence determined, trpA(UGA211) gave rise to two previously unidentified types of missense revertant. The first type was expected, namely trpA(CGA211), the result of a base substitution event. The other type, representing approximately 1% of the missense revertants, was unexpected on the basis of single base substitutions and an understanding of which amino acids are functional at alpha chain position 211. It was found to be the result of a 21 base-pair deletion of a region containing codon 211. The tryptophan-independent revertants of both position 234 nonsense mutants occurred at a fre-quew~cy of approximately 2 per 109 viable cells. They were identical in that they both resulted from a 3 base-pair deletion~ namely deletion of the chain-terminating codon at position 234. One of them, however, also displayed an A instead of the normal G in the third position of codon 235. The revertants were characterized according to growth in different media and tryptophan synthetase assays performed on crude extracts. These types of mutants should prove interesting and important for the elucidation of alpha chain structure-function relationships, for insight into the assembly and interaction of subunits in this model muitienzyme complex, and for the study of mechanisms by which deletions can be generated. tryptophan synthetase / 3 bp deletion / 21 bp deletion / multienzyme complex
Proceedings of the National Academy of Sciences of the United States of America, Jun 1, 1988
We have isolated an unusual codon-specific translational suppressor in Escherichia coli. The supp... more We have isolated an unusual codon-specific translational suppressor in Escherichia coli. The suppressor resulted from a spontaneous mutation in a chromosomal gene during a selection for suppressors of the auxotrophic nonsense mutation trpA(UGA211). The suppressor allows readthrough of UGA mutations at two positions in trpA and at two sites in bacteriophage T4. It does not, however, suppress amber (UAG) or ochre (UAA) mutations that were tested in both genomes, some of which were at the same positions as the suppressible UGA mutations. The suppressor also does not allow mistranslation of the UGA-related tqpA missense mutations UGG at
Proceedings of the National Academy of Sciences, 1991
Site-directed mutagenesis was performed on a sequence motif within the 3' major domain of Esc... more Site-directed mutagenesis was performed on a sequence motif within the 3' major domain of Escherichia coli 16S rRNA shown previously to be important for peptide chain termination. Analysis of stop codon suppression by the various mutants showed an exclusive response to UGA stop signals, which was correlated directly with the continuity of one or the other of two tandem complementary UCA sequences (bases 1199-1204). Since no other structural features of the mutated ribosomes were hampered and the translation initiation and elongation events functioned properly, we propose that a direct interaction occurs between the UGA stop codon on the mRNA and the 16S rRNA UCA motif as one of the initial events of UGA-dependent peptide chain termination. These results provide evidence that base pairing between rRNA and mRNA plays a direct role in termination, as it has already been shown to do for initiation and elongation.
Proceedings of the National Academy of Sciences, 1983
In a previous publication, an unusual UGG-reading missense suppressor caused by insertion of an e... more In a previous publication, an unusual UGG-reading missense suppressor caused by insertion of an extra adenylate residue in the anticodon loop of an Escherichia coli glycine tRNA was described. In this study, we provide in vivo evidence that the additional nucleotide causes an "anticodon shift" by one nucleotide in the 3' direction and that the "new" anticodon can explain the unanticipated coding properties of the suppressor. We converted the UGG suppressor with ethyl methanesulfonate, a base-substitution mutagen, to suppressors that read codons related to UGG by a single base change. Sequence analysis of each mutant tRNA revealed that its mutational alteration was an anticipated base change in one of the three nucleotides of the "new" anticodon. Although the new suppressors read codons beginning with A or U, the mutant tRNAs lack the customary hypermodified nucleosides on the 3' side of the anticodon. As determined on the basis of their in vivo ...
Beginning with a missense suppressor tRNA and a nonsense suppressor tRNA, both in Escherichia col... more Beginning with a missense suppressor tRNA and a nonsense suppressor tRNA, both in Escherichia coli and each containing an extra nucleotide in the anticodon loop, we generated new suppressors in vivo by spontaneous deletion of specific nucleotides from the anticodon loop. In one experiment, the new suppressor was generated by a double mutational event, base substitution and nucleotide deletion. A novel ochre suppressor is also described. It is very efficient in nonsense suppression but has no ms2i6 modification of the A residue on the 3' side of the anticodon. The results have important implications for tRNA structure-function relationships, tRNA recognition by tRNA-modifying enzymes, mechanisms of deletion mutation, and tRNA evolution.
Previous studies have demonstrated many amino acid and codon substitutions at position 211 of the... more Previous studies have demonstrated many amino acid and codon substitutions at position 211 of the alpha chain of tryptophan synthetase of Escherichia coli. In order to extend our studies on suppressor tRNAs and, in general, on accuracy in the translation of genetic information, we have devised specific selections for sense and nonsense codons corresponding to alpha chain position 234. Using codon-specific suppressors and selection for missense revertants of nonsense mutations, we have brought to 18 (from six) the number of codons at 234. This number includes all three nonsense codons and ten (of the remaining 15) that code for nonfunctional amino acids at that position. Furthermore, by adding to these results the testing of the suppressibility of trpA(234) nonsense mutations by suppressor tRNAs of known aminoacylation specificity, we have increased to 12 (from five) the number of amino acids whose functionality at position 234 is known. The existence of trpA mutants with so many amino acid substitutions at two positions in the tryptophan synthetase alpha chain should prove helpful for structure-function studies of that protein. Furthermore, the availability, at both positions, of at least 12 codons (including the three nonsense codons) that result in a nonfunctional alpha chain allows the selection of mutations that affect the suppression of a given codon as it occurs in two different mRNA contexts. Studies with such mutants should help elucidate the role in translational accuracy of mRNA secondary structure, tRNA-tRNA interactions on the ribosome, and interactions of the ribosome with tRNA, mRNA, and, in the case of nonsense suppression, peptidyl release factors.
RNA sites that contain unpaired or mismatched nucleotides can be interaction sites for other macr... more RNA sites that contain unpaired or mismatched nucleotides can be interaction sites for other macromolecules. C1054, a virtually universally conserved nucleotide in the 16 S (small subunit) ribosomal RNA of Escherichia coli, is part of a highly conserved bulge in helix 34, which has been located at the decoding site of the ribosome. This helix has been implicated in several translational events, including peptide chain termination and decoding accuracy. Here, we observed interesting differences in phenotype associated with the three base substitutions at, and the deletion of, nucleotide C1054. The phenotypes examined include suppression of nonsense codons on different media and at different temperatures, lethality conditioned by temperature and level of expression of the mutant rRNA, ribosome pro®les upon centrifugation through sucrose density gradients, association of mutant 30 S subunits with 50 S subunits, and effects on the action of tRNA suppressor mutants. Some of our ®ndings contradict previously reported properties of individual mutants. Particularly notable is our ®nding that the ®rst reported 16 S rRNA suppressor of UGA mutations was not a C1054 deletion but rather the base substitution C1054A. After constructing ÁC1054 by site-directed mutagenesis, we observed, among other differences, that it does not suppress any of the trpA mutations previously reported to be suppressed by the original UGA suppressor. In general, our results are consistent with the suggestion that the termination codon readthrough effects of mutations at nucleotide 1054 are the result of defects in peptide chain termination rather than of decreases in general translational accuracy. The phenotypic heterogeneity associated with different mutations at this one nucleotide position may be related to the mechanisms of involvement of this nucleotide, the two-nucleotide bulge, and/or helix 34 in particular translational events. In particular, previous indications from other laboratories of conformational changes associated with this region are consistent with differential effects of 1054 mutations on RNA-RNA or RNA-protein interactions. Finally, the association of a variety of phenotypes with different changes at the same nucleotide may eventually shed light on speculations about the coevolution of parts of ribosomal RNA with other translational macromolecules.
After our first observation of codon context effects in missense suppression ( Murgola & ... more After our first observation of codon context effects in missense suppression ( Murgola & Pagel , 1983), we measured the suppression of missense mutations at two positions in trpA in Escherichia coli. The suppressible codons in the trpA messenger RNA were the lysine codons, AAA and AAG, and the glutamic acid codons, GAA and GAG. The mRNA sites of the codons correspond to amino acids 211 and 234 of the trpA polypeptide, positions at which glycine is the wild-type amino acid. Our data demonstrated codon context effects with both pairs of codons. The results indicate that suppression of AAA and AAG by mutant lysine transfer RNAs was more efficient at 211 than at 234, whereas suppression of GAA and GAG by two different mutant glycine tRNAs was more efficient at 234 than at 211. In general, the context effects were more pronounced with GAG and AAG than with GAA and AAA. (In some instances it appeared that suppression of GAA or AAA at a given position was more effective than suppression of GAG or AAG.) By contrast, no context effects were observed with a glyT suppressor of AAA and AAG, a glyT GAA/G-suppressor, and a glyU suppressor of GAG. Our observation of this phenomenon in missense suppression demonstrates that codon context can affect polypeptide elongation and that the effects can be different depending on the codons and tRNAs examined. It is suggested that tRNA-tRNA interaction on the ribosome is involved in the observed context effects.
A modified assay has been devised for the physiological reaction, indole-3-glycerol phosphate to ... more A modified assay has been devised for the physiological reaction, indole-3-glycerol phosphate to Trp, of the enzyme tryptophan synthetase. The assay may be applied to crude bacterial extracts, and is based on the measurement of incorporation of radioactivity from [3H]Ser into Trp. Comparison with previous colorimetric assays indicates an improvement in sensitivity of about 30-fold, and advantages in terms of sample economy and simplified manipulation.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1990
The base sequence around nonsense codons affects the efficiency of nonsense codon suppression. Pu... more The base sequence around nonsense codons affects the efficiency of nonsense codon suppression. Published data, comparing different nonsense sites in a mRNA, implicate the two bases downstream of the nonsense codon as major determinants of suppression efficiency. However, the results we report here indicate that the nature of the contiguous upstream codon can also affect nonsense suppression, as can the third (wobble) base of the contiguous downstream codon. These conclusions are drawn from experiments in which the two Set codons UCU233 and UCG235 in a nonsense mutant form (UGA234) of the trpA gene in Escherichia coil have been replaced with other Ser codons by site-directed mutagenesis. Suppression of these trpA mutants has been studied in the presence of a UGA nonsense suppressor derived from glyT. We speculate that the non-site-specific effects of the two adjacent downstream bases may be largely at the level of the termination process, whereas more site-specific or codon-specific effects may operate primarily on the activity of the suppressor tRNA.
To screen for ribosomal RNA mutants defective in peptide chain termination, we have been looking ... more To screen for ribosomal RNA mutants defective in peptide chain termination, we have been looking for rRNA mutants that exhibit different patterns of suppression of nonsense mutations and that do not suppress missense mutations at the same positions in the same reporter gene. The rRNA mutations were induced by segment-directed randomly mutagenic PCR treatment of a cloned rrnB operon, followed by subcloning of the mutagenesis products and transformation of strains containing different nonsense mutations in the Escherichia coli trpA gene. To date, we have repeatedly obtained only two small sets of mutations, one in the 3′ domain of 16S rRNA, at five nucleotides out of the 610 mutagenized (two in helix 34 and three in helix 44), and the other in 23S rRNA at only four neighboring nucleotide positions (in a highly conserved hexanucleotide loop) within me 1.4 kb mutagenized segment. There is variety, however, in the suppression patterns of the mutants, ranging from suppression of UAG or UG...
m This paper describes a novel mechanism for reversion of nonsense mutations in the trpA gene of ... more m This paper describes a novel mechanism for reversion of nonsense mutations in the trpA gene of F~cherichia coli. This mechanism, deletion of the nonsense codon, was discovered in the course of select _~ for missense revertants of trpA(UGA211) and for catalytically active tryptophan synthetase alpha chain revertants of trpA(UAA234) and trpA(UAG234). Each type of revertant trpA was cloned and its DNA sequence determined, trpA(UGA211) gave rise to two previously unidentified types of missense revertant. The first type was expected, namely trpA(CGA211), the result of a base substitution event. The other type, representing approximately 1% of the missense revertants, was unexpected on the basis of single base substitutions and an understanding of which amino acids are functional at alpha chain position 211. It was found to be the result of a 21 base-pair deletion of a region containing codon 211. The tryptophan-independent revertants of both position 234 nonsense mutants occurred at a fre-quew~cy of approximately 2 per 109 viable cells. They were identical in that they both resulted from a 3 base-pair deletion~ namely deletion of the chain-terminating codon at position 234. One of them, however, also displayed an A instead of the normal G in the third position of codon 235. The revertants were characterized according to growth in different media and tryptophan synthetase assays performed on crude extracts. These types of mutants should prove interesting and important for the elucidation of alpha chain structure-function relationships, for insight into the assembly and interaction of subunits in this model muitienzyme complex, and for the study of mechanisms by which deletions can be generated. tryptophan synthetase / 3 bp deletion / 21 bp deletion / multienzyme complex
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Papers by Kathryn Hijazi