Analysis and manipulation of yeast mitochondrial genes.
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Cloning, Molecular↗
Biomedical subjects
L S Folley
Publications and source records attributed to L S Folley.
Site-directed mutagenesis of a Saccharomyces cerevisiae mitochondrial translation initiation codon.
We have used a generally applicable strategy for gene replacement in yeast mitochondria to mutate the translation initiation codon of the COX3 gene from AUG to AUA. The mutation, cox3-1, substantially reduced, but did not eliminate, translation of cytochrome c oxidase subunit III (coxIII). Strains bearing the mutation exhibited a leaky (partial) nonrespiratory growth phenotype and a reduced incorporation of radiolabeled amino acids into coxIII in vivo in the presence of cycloheximide. Hybridization experiments demonstrated that the mutation had little or no effect on levels of the COX3 mRNA. Residual translation of the cox3-1 mutant mRNA was dependent upon the three nuclearly coded mRNA-specific activators PET494, PET54 and PET122, known from previous studies to work through a site (or sites) upstream of the initiation codon to promote translation of the wild-type mRNA. Furthermore, respiratory growth of cox3-1 mutant strains was sensitive to decreased dosage of genes PET494 and PET122 in heterozygous mutant diploids, unlike the growth of strains carrying wild-type mtDNA. Some residual translation of the cox3-1 mRNA appeared to initiate at the mutant AUA codon, despite the fact that the 610-base 5'-mRNA leader contains numerous AUA triplets. We conclude that, while AUG is an important component of the COX3 translation initiation site, the site probably is also specified by other sequence or structural features.
Codon↗
Codon contexts from weakly expressed genes reduce expression in vivo.
Nucleotides that neighbor codons in Escherichia coli genes are highly non-random. Furthermore, these context biases are stronger and extend farther from the codon in weakly expressed than in highly expressed genes. We therefore suggested that codon contexts are selected to reduce gene expression levels. We now compare the expression levels of lacZ genes containing two specific coding sequences (context inserts). One context insert represents contexts seen in weakly expressed genes (low variant); the other represents contexts seen in highly expressed genes (high variant). The two variants have identical nucleotide and codon compositions, and encode the same protein. A permutation of four nucleotides, which changes eight codon:codon interfaces of 1043, comprises the only difference between the high and low context variant genes. In three different lacZ mRNAs, the low variant was expressed at a level significantly below that of the high variant. This context effect depends entirely on translation of the contexts in the correct frame; its magnitude depends in part on the placement of other features (e.g. transcriptional pauses and terminators, or perhaps other slow codons or contexts) in the mRNAs. Changing the ribosome density on the message by changing the ribosome binding site distinguishes between dropoff, interference and polarity, three fundamentally different types of models for the context effect. The expression difference between context variants is eliminated by both increases and decreases in the ribosome initiation frequency, as uniquely predicted by the polarity model. In fact, data from all constructions are accommodated by a model in which slow translation of the low context insert increases rho-dependent transcriptional termination within the test gene. The data suggest that the rates of translational initiation and elongation are poised with respect to the rate of transcriptional elongation so that all are influential in setting the expression level of wild-type lacZ. We conclude that context-induced polarity will exist in genes wherever low and reproducible gene product levels have been selected.
Amino Acid Sequence↗
Sense codons are found in specific contexts.
The sequence environment of codons in structural genes has been investigated statistically, using computer methods. A set of Escherichia coli genes with abundant products was compared with a set having low gene product levels, in order to detect potential differences associated with expression. The results show striking non-randomness in the nucleotides occurring near codons. These effects are, unexpectedly, very much larger and more homogeneous among the genes with rare products. The intensity of effects in weakly expressed genes suggests that such non-random sequence environments decrease expression. In the weakly expressed set of genes, the 5' neighbor of a codon, and all positions of the 3' neighbor codon are biased. In the highly expressed genes, the first nucleotide of the next codon is a uniquely affected site. The distribution of non-randomness in weakly expressed genes suggests that sequence bias is primarily due to a constraint acting directly on the secondary or tertiary structure of the codon/anticodon. In highly expressed genes, the observed bias suggests an interaction between the codon/anticodon and a site outside the codon/anticodon. Much of the tendency to non-random near-neighbor sequences in weakly expressed genes can be ascribed to a correlation between nearby nucleotides and the wobble nucleotide of the codon, despite the fact that selection of such correlations will alter the amino acid sequence. The favored pattern, in genes expressed at low level, is R YYR or Y RRY. R indicates purine, Y indicates pyrimidine; the space is the boundary between codons. It seems likely that this preference for nearby sequences is the physical basis of the genetic context effect. Under this assumption such sequence biases will affect expression. On this basis, we predict new sites for contextual mutations which decrease expression, and suggest strategy for the design of messages having optimal translational activity.
Amino Acids↗