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J A Gallant

Publications and source records attributed to J A Gallant.

At least 19 recordsLinked to original sources
PubMed1998

Ribosomes can slide over and beyond "hungry" codons, resuming protein chain elongation many nucleotides downstream.

In cells subjected to moderate aminoacyl-tRNA limitation, the peptidyl-tRNA-ribosome complex stalled at the "hungry" codon can slide well beyond it on the messenger RNA and resume translation further downstream. This behavior is proved by unequivocal amino acid sequence data, showing a protein that lacks the bypassed sequence encoded between the hungry codon and specific landing sites. The landing sites are codons cognate to the anticodon of the peptidyl-tRNA. The efficiency of this behavior can be as high as 10-20% but declines with the length of the slide. Interposition of "trap" sites (nonproductive landing sites) in the bypassed region reduces the frequency of successful slides, confirming that the ribosome-peptidyl-tRNA complex passes through the untranslated region of the message. This behavior appears to be quite general: it can occur at the two kinds of hungry codons tested, AUA and AAG; the sliding peptidyl-tRNA can be any of three species tested, phenylalanine, tyrosine, or leucine tRNA; the peptidyl component can be either of two very different peptide sequences; and translation can resume at any of the three codons tested.

Amino Acid Sequence↗
PubMed1994

The function of a ribosomal frameshifting signal from human immunodeficiency virus-1 in Escherichia coli.

A 15-17 nucleotide sequence from the gag-pol ribosome frameshift site of HIV-1 directs analogous ribosomal frameshifting in Escherichia coli. Limitation for leucine, which is encoded precisely at the frameshift site, dramatically increased the frequency of leftward frameshifting. Limitation for phenylalanine or arginine, which are encoded just before and just after the frameshift, did not significantly affect frameshifting. Protein sequence analysis demonstrated the occurrence of two closely related frameshift mechanisms. In the first, ribosomes appear to bind leucyl-tRNA at the frameshift site and then slip leftward. This is the 'simultaneous slippage' mechanism. In the second, ribosomes appear to slip before binding aminoacyl-tRNA, and then bind phenylalanyl-tRNA, which is encoded in the left-shifted reading frame. This mechanism is identical to the 'overlapping reading' we have demonstrated at other bacterial frameshift sites. The HIV-1 sequence is prone to frame-shifting by both mechanisms in E. coli.

Amino Acid Sequence↗
PubMed1994

Phosphorolytic error correction during transcription.

Escherichia coli DNA-directed RNA polymerase is shown to contain a novel phosphorolytic error correction activity which removes erroneous nucleotides, as rNDPs, from the 3'-end of the growing transcript. The activity we describe is biochemically similar to polynucleotide phosphorylase (PNP), yet in contrast to PNP is activated by Mn2+. We demonstrate that the activity, which is mediated by Pi, is dependent on the presence of an incorrectly incorporated nucleotide at the leading 3'-end of the transcript. The correction activity we describe exhibits a 4 x 10(4)-fold preference for the excision of incorrect nucleotides from the transcript. These findings suggest the possibility that RNA phosphorolysis may play a critical role in the process of transcriptional proofreading.

Bacterial Proteins↗
PubMed1993

On the role of the P-site in leftward ribosome frameshifting at a hungry codon.

Previous work characterized ribosomal frameshifting within the sequence C UUC AAG provoked by lysyl-tRNA limitation. The ribosome frameshift is one base to the left of the AAG lysine codon, as shown by dotted overlining above. We now show that the frequency of this leftward ribosome frameshift is strongly influenced by the identity of the bases two, three and four positions to the left of the actual frameshift site. The nature of these influences coincides exactly with the possibilities of base-pairing between the sequence and the anticodon of the P-site peptidyl-tRNA when shifted one base to the left just upstream of the frameshift site. We conclude that a peptidyl shift in the P-site is intimately involved in leftward frameshifting in the adjacent A site when it codes for an aminoacyl-tRNA in short supply.

Amino Acid Sequence↗
PubMed1992

Leftward ribosome frameshifting at a hungry codon.

Previous experiments have shown that limitation for certain aminoacyl-tRNA species results in phenotypic suppression of a subset of frameshift mutant alleles, including members in both the (+) and (-) incorrect reading frames. Here, we demonstrate that such phenotypic suppression can occur through a ribosome reading frame shift at a hungry AAG codon calling for lysyl-tRNA in short supply. Direct amino acid sequence analysis of the product and DNA sequence manipulation of the gene demonstrate that the ribosome frameshift occurs through a movement of one base to the left, so as to decode the triplet overlapping the hungry codon from the left or 5' side, followed by continued normal translation in the new, shifted reading frame.

Amino Acid Sequence↗
PubMed1992

Context rules of rightward overlapping reading.

We have investigated the mechanism and sequence context rules governing ribosome frameshifting promoted by aminoacyl-tRNA limitation. In the case of one shifty sequence, frameshifting promoted by lysyl-tRNA limitation occurs at the sequence AAG C and is due to rightward movement of the ribosome so as to read the AGC triplet overlapping the hungry codon from the right. The frequency of this event is unaffected by sequence elements more than three bases to the left (upstream) or two bases to the right (downstream) of the hungry codon, and only slightly affected by the identity of the base two bases to the right. It is strongly affected by the base immediately to the right of the hungry codon, which becomes the wobble base of the shifted triplet; and by the third base of the hungry codon, even though the two synonyms (AAG and AAA) call for the same aminoacyl-tRNA; and by the identity of the base immediately to the left of the hungry codon. The latter result suggests that the aminoacyl-tRNA in the P site affects the maintenance of reading frame at the adjacent A site of the ribosome. However, the DNA sequence makes it seem unlikely that the P-site tRNA shifts to the right in concert with the A-site tRNA, a mechanism that can account for leftward frameshifting (in the opposite direction) in retroviral translation. The specificity of sequence determinants of leftwing versus rightwing frameshifting is discussed.

Amino Acid Sequence↗
PubMed1991

The role of RNA polymerase in transcriptional fidelity.

The overall transcription of DNA has previously been demonstrated to proceed at extremely high levels of accuracy. We review the evidence that the process of transcription is subject to proof-reading in the Hopfield sense. In addition, we speculate that the proof-reading activity associated with transcription is subject to cyclical phase transitions. That is, during periods of low processivity associated with initiation, RNA synthesis is relatively imprecise. The transition to the elongation phase of RNA synthesis, characterized by a shift to high processivity, is accompanied by enhanced proof-reading. A model for the damping of transcriptional errors, based on a PPi-mediated processive pyrophosphorolysis reaction, is discussed in terms of pausing during transcription.

Bacterial Proteins↗
PubMed1989

Transcriptional proofreading in Escherichia coli.

A novel transcriptional proofreading mechanism associated with the beta-subunit of wild-type RNA polymerase from Escherichia coli is suggested from the following data. The purified holoenzyme contains an NTPase activity which specifically converts noncognate NTPs to their corresponding NDP in a template-dependent manner during in vitro transcription of synthetic single- and double-stranded templates. In contrast, purified enzyme from an rpoB mutant which shows increased transcriptional error lacked template-dependent NTP hydrolytic activity. The NTP hydrolytic activity of wild-type enzyme was critically dependent on the integrity of the initiation complex, and required continued transcriptional elongation. Transcription and translation of the lacZ gene proceeded 17% faster in the mutant than in its wild-type parent. These results are discussed in terms of a proofreading model in which the rate of transcription is limited by proofreading events that involve recognition and hydrolysis of noncognate NTPs before they can be misincorporated into RNA.

DNA-Directed RNA Polymerases↗
PubMed1986

An RNA polymerase mutant with reduced accuracy of chain elongation.

A new Escherichia coli RNA polymerase mutant was isolated which exhibited reduced accuracy of chain elongation in vivo and in vitro. The novel isolation procedure consisted of simultaneous selection for rifampicin resistance and screening for increased leakiness of an early, strongly polar nonsense mutation of lacZ, one of a special class of mutations whose leakiness reflects mainly transcriptional rather than translational errors. The spontaneous mutant thus isolated displayed a 3-4-fold increase in the leakiness of two different lacZ mutations of this class. Transduction analysis indicated that a single mutation, mapping in or very near the rpoB gene for the beta subunit of RNA polymerase, conferred both rifampicin resistance and increased nonsense leakiness. In an in vitro fidelity assay, homogeneous RNA polymerases from the mutant and parent strains exhibited error rates of 1/0.90 X 10(5) and 1/2.0 X 10(5), respectively, for the poly[d(A-T)] X poly[d(A-T)]-directed misincorporation of noncomplementary GMP. These error rates were verified by product analyses which further revealed that GMP was misincorporated in place of AMP in the synthesis of poly[r(A-U)]. The error rate of wild-type K12 RNA polymerase from a different source was 1/2.0 X 10(5), while that of a hybrid RNA polymerase, containing mutant core enzyme and wild-type sigma subunit, was 1/0.64 X 10(5). These error rates confirmed the selection of a transcriptional accuracy mutant. The error frequencies observed are much lower than those reported in other in vitro assays. The safeguards used to avoid artifactually enhanced misincorporation, and to thereby quantitate lower error rates, are discussed.

DNA-Directed RNA Polymerases↗
PubMed1986

Uncharged tRNA error damping model.

Uncharged tRNA is known to bind to the ribosome in a codon-specific fashion. In this way, cognate uncharged tRNA competes with non-cognate aminoacyl-tRNA. If uncharged tRNA can be aminoacylated while on the ribosome, this will damp errors due to aminoacyl-tRNA imbalance. Kinetic analysis shows that this scheme reduces errors at 'hungry' codons considerably more effectively than J. Ninio's accuracy tuner model; for example, a 10-fold decrease in cognate aminoacyl-tRNA elicits only a 10% increase in the error frequency.

Binding, Competitive↗
PubMed1986

Frameshift suppression in aminoacyl-tRNA limited cells.

Under certain conditions aminoacyl-tRNA limitation can phenotypically suppress frameshift alleles. The observed suppression is due to an increase in abnormal translocation of ribosomes translating codons that have a short supply of aminoacyl-tRNA. The rIIB frameshift alleles of bacteriophage T4 are used here to pinpoint the sites of ribosome frameshifting caused by these hypothetical decoding errors. The data indicate that not all hungry codons are associated with abnormal translocation, only a relatively small subset. Analysis of the hungry codons which are associated with ribosome frameshifting points to the existence of severe context effects determining the shiftiness of these codons.

Alleles↗
PubMed1984

Genetic screen for cloned release factor genes.

Nonsense suppression reflects competition between a nonsense suppressor tRNA and a translational release factor. This provides a simple way to screen for release factor genes cloned into a multicopy plasmid. We have confirmed that the expected competition occurs with the gene for release factor 2, cloned by C.T. Caskey et al. (C.T. Caskey, W.C. Forrester, W. Tate, and C.D. Ward, J. Bacteriol. 158:365-368, 1984), and used it to clone the gene for release factor 1.

Cloning, Molecular↗
PubMed1983

The glucose effect in Bacillus subtilis.

An analysis of the glucose downshift mechanism in Bacillus subtilis has shown that the depression of catabolic enzymes characteristic of the 'glucose effect' includes isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase. Additionally, phosphofructokinase undergoes what appears to be a reversible modification regulated by glucose transport.

Bacillus subtilis↗
PubMed1983

Bacillus subtilis relG mutant: defect in glucose uptake.

A previously described Bacillus subtilis mutant which exhibits a relaxed phenotype after glucose limitation (relG) has been characterized further. Analysis of this mutant and of the downshift mechanism in B. subtilis has shown that the primary defect lies in glucose uptake.

Bacillus subtilis↗
PubMed1982

A new relaxed mutant of Bacillus subtilis.

A new relaxed mutant of Bacillus subtilis was isolated by screening Rifr clones for alterations in stringent control. The Rifr relaxed mutant which is described was found to contain a second-site mutation conferring a relaxed response to an energy source downshift and was partially relaxed after amino acid starvation. The new rel locus, called relG, was distinct from the two other known rel loci in B. subtilis, relA, and relC.

Amino Acids↗
PubMed1981

A model of clonal attenuation.

Two formal models of clonal attenuation [Kirkwood, T. B. L. & Holliday, R. (1975) J. Theor. Biol. 53, 481-496; Shall, S. & Stein, W. D. (1979) J. Theor. Biol. 76, 219-231] are considered in the light of recent data on the changing distribution of replicative potential among individual cultured fibroblasts on subcloning. The experimental data [Smith, J. R., Pereira-Smith, O. & Good, P. I. (1977) Mech. Ageing Dev. 6, 283-286] are shown to contradict both models. A new model, compatible with the subcloning data, is proposed. This model involves a gradual increase in the probability of commitment during cell growth in culture and a small number (about seven) of divisions following commitment. The gradual increase in commitment probability is shown to be compatible with the gradual accumulation of a gene product subject to autogenous regulation.

Cell Differentiation↗
PubMed1980

Regulation of ribosomal and transfer RNA synthesis by guanosine 5'-diphosphate-3'-monophosphate.

Permeabilized cells of Escherichia coli were used to examine the effect on RNA synthesis of guanosine 5'-diphosphate-3'-monophosphate (ppGp). Electrophoretic and hybridization analysis of bulk RNA demonstrated that ppGp selectively reduced the accumulation of both ribosomal and transfer RNAs. The experiments further suggested that the observed reduction in stable RNA accumulation resulted from a reduction in stable RNA chain initiation rather than an effect on elongation, processing, or maturation of stable RNA transcripts. In contrast, the expression of the lac Z gene was not affected by ppGp. These results suggest that ppG, a nucleotide produced in E. coli during the stringent response, could play a direct role in the stringent regulation of stable RNA synthesis.

Escherichia coli↗