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J F Curran

Publications and source records attributed to J F Curran.

8 recordsLinked to original sources
PubMed1991

Effects of the nucleotide 3' to an amber codon on ribosomal selection rates of suppressor tRNA and release factor-1.

Rates of ribosomal selection of both release factor 1 (RF1) and a suppressor tRNA (Su7C33) were studied at an amber codon at which the 3' neighbor was permuted. Rates of RF1 selection vary 2.6-fold among contexts. The 3' neighbor-dependent variation of RF1 action correlates very strongly with the non-random frequencies of 3' neighbors at UAG terminators (r = 0.97), which argues that the rate of RF1 selection is an important determinant 3' neighbor choice at termination codons. The data are consistent with a model for RF1 selection in which RF1 makes a specific contact(s) to the 3' neighbor and that this interaction is most favorable to uridylic acid. Measured rates of Su7C33 selection vary fivefold among 3' contexts. We also develop a method to calculate rates of selection for other suppressors, based on the assumption that rates of RF1 selection at each 3' context can be generalized to other sites that have the same 3' neighbor. Rates for various suppressors appear to vary from two- to fivefold depending on the 3' neighbor. Generally, the rate of selection of suppressors at different contexts correlates with the stacking strength of the 3' neighbor as measured in vitro. The two- to fivefold range of 3' neighbor effects on rate of aminoacyl-tRNA selection is greater than that previously observed within sets of codons read by the same tRNA. It is suggested that the choice of codons to achieve favorable contexts may be more important than the choice of a common codon at some message sites.

Base Sequence↗
PubMed1989

Rates of aminoacyl-tRNA selection at 29 sense codons in vivo.

We have placed aminoacyl-tRNA selection at individual codons in competition with a frameshift that is assumed to have a uniform rate. By assaying a reporter in the shifted frame, relative rates for association of the 29 YNN codons and their cognate aminoacyl-tRNAs were obtained during logarithmic growth in Escherichia coli. For five codons, three beginning with C and two with U, these relative rates agree with relative in vitro rates for elongation factor Tu-mediated aminoacyl-tRNA binding to ribosomes and subsequent GTP hydrolysis. Therefore, the frameshift assay probably measures this process in vivo. Observed rates for aminoacyl-tRNA selection span a 25-fold range. Therefore, the time required to transit different codons in vivo probably differs substantially. Codons very frequently used in highly expressed genes generally select aminoacyl-tRNAs more quickly than do rarely used codons. This suggests that speed of aminoacyl-tRNA selection is a significant factor determining biased use of synonymous codons. However, the preferential use of codons appears to be marked only for codons with the highest rates of aminoacyl-tRNA selection. Rapid selection in vivo is usually effected by elevation of the tRNA concentration for codons with moderate intrinsic speed (rate constant), not by choosing intrinsically fast codons. Despite a preference for high rate, there are quickly translated codons that are not commonly used, and common codons that are translated relatively slowly. Other factors are therefore more important than speed for some codons. Strong preference for rapid aminoacyl-tRNA selection is not observed in weakly expressed genes. Instead, there is a slight preference for slower aminoacyl-tRNA selection. The rate of aminoacyl-tRNA selection by a YNC codon is always greater than the rate of the corresponding YNU codon even though in many YNC/U pairs both codons react with the same elongation factor Tu/GTP/aminoacyl-tRNA complex. Thus, for these tRNAs, the differences between in vivo rate constants of tRNAs are dependent on the nature of anticodon base-pairing. However, no more general relationship is evident between codon/anticodon composition and rate of aminoacyl-tRNA selection. The frameshift method can be extended to all codons.

Base Sequence↗
PubMed1988

Use of tRNA suppressors to probe regulation of Escherichia coli release factor 2.

It has been suggested that Escherichia coli release factor 2 (RF-2) translation is autoregulated. Mature RF-2 protein can terminate its own nascent synthesis at an intragenic, in-phase UGA codon, or alternatively, a +1 frameshift can occur that leads to completion of the RF-2 polypeptide. Translational termination presumably increases with RF-2 concentration, providing negative regulatory feedback. We now show, in lacZ/RF-2 fusions, that translation of a UAG codon at the position of the UGA competes with frameshifting, which proves one postulate of the translational autoregulatory model. We also identify a nearby sequence that is required for high-frequency frameshifting and suggest a constraint for the codon preceding the shift point. Both these sequences are incorporated into a model for frameshifting. Our measurements allow us to compute the relative rates in vivo of these reactions: release factor action, frameshifting and tRNA selection at an amber codon.

Codon↗
PubMed1987

Reading frame selection and transfer RNA anticodon loop stacking.

Messenger RNA's are translated in successive three-nucleotide steps (a reading frame), therefore decoding must proceed in only one of three possible frames. A molecular model for correct propagation of the frame is presented based on (i) the measured translational properties of transfer RNA's (tRNA's) that contain an extra nucleotide in the anticodon loop and (ii) a straightforward concept about anticodon loop structure. The model explains the high accuracy of reading frame maintenance by normal tRNA's, as well as activities of all characterized frameshift suppressor tRNA's that have altered anticodon loops.

Anticodon↗
PubMed1986

Base substitutions in the tRNA anticodon arm do not degrade the accuracy of reading frame maintenance.

We have examined the activities of a set of 34 site-directed mutants of tRNA Su7 for their ability to shift reading frame during translation of amber codons in vivo. The set includes variants at every position in the distal three base pairs of the anticodon stem and saturates the anticodon loop, with the exception of the anticodon itself. Most anticodon-stem mutations were made pairwise to preserve the secondary structure of that region. Variants of the Hirsh (A24) coding alteration were also tested. The mutations have varied and often dramatic effects on the ability of Su7 to act in translation, which indicates that they cause distortions of the codon-anticodon complex. However, none of the tested mutations affects the intrinsic accuracy of translocation, which we show to be very high. These results suggest that translocation must be independent of the conformational detail of the codon-anticodon complex and stand in contrast to frameshifts that occur when tRNAs misread codons. We suggest that when the tRNA is properly paired to the codon, translocation proceeds normally. Thus, we conclude that selection of a cognate tRNA ensures highly accurate reading frame maintenance. As a corollary, inefficient amber suppressors are not inefficient because they frameshift. Instead, they are likely to fail because a release factor translates the amber codon.

Anticodon↗
PubMed1985

Cloning and mapping of the SPO1 genome.

Many of the XbaI, EcoRI, KpnI, and BglII fragments of bacteriophage SPO1, accounting for about 65% of the genomic sequences, were cloned in Bacillus subtilis. Four of the EcoRI fragments were specifically refractory to cloning in both Escherichia coli and B. subtilis, probably because of expression of deleterious genes carried on the SPO1 fragments. To permit complete identification of the regions cloned, the SPO1 restriction map has been extended to include the XbaI fragments and the previously unmapped KpnI fragments. Markers for 26 of the 39 known genes have been located on specific cloned fragments, permitting more precise determination of the positions of most of the genes. One cloned SPO1 fragment was inhibitory to SPO1 development.

Bacillus subtilis↗
PubMed1985

Transcription of Bacillis subtilis plasmid pBD64 and expression of bacteriophage SPO1 genes cloned therein.

Plasmid pBD64, a vector which is useful for cloning in Bacillis subtilis (T. J. Gryczan, A. G. Shivakumar, and D. Dubnau (1980), J. Bacteriol. 141, 246-253), has at least three substantial transcription units. Two of these include the single EcoRI, XbaI, and BamHI sites, while the other includes the single BglII site. Each of these transcripts was synthesized in the counterclockwise direction, relative to the pBD64 restriction map. No transcripts were detected in the opposite direction. Infection by bacteriophage SPO1 caused a substantial decrease in each of these transcripts. No new pBD64 transcripts were detected during SPO1 infection. Various SPO1 genes, cloned at several of these pBD64 sites, were tested for expression by observing their capacity to complement SPO1 mutants. Several middle and late genes were expressed substantially, regardless of the orientation in which the fragments were inserted. Since transcription from the vector could cause expression only in one orientation, this argues that the necessary transcription originated at SPO1 promoters, and, thus, that SPO1 middle and late promoters can be active in thymine-containing DNA.

Bacillus subtilis↗