Biomedical subjects

J A Gallant

Publications and source records attributed to J A Gallant.

13 recordsLinked to original sources

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↗

PubMed1979