Post-Transcriptional Control of Gene Expression : mRNA Degradation and Translational Regulation

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1. Identification of the prfC gene encoding peptide-chain-release factor 3 of Escherichia coli : The termination of protein synthesis in bacteria requires two codon-specific polypeptide release factors, RF-1 and RF-2. A third factor, RF-3, that stimulates the RF-1 and RF-2 activities, was originally identified in E.coli, but it has received little attention since the 1970's. To search for the gene encoding RF-3, we selected novel nonsense-suppressor mutations by random insertion mutagenesis. One of these mutations, named tos-1, mapped to the 99.2 min region on the E. coli chromosome and suppressed all three stop codons. Complementation studies and analyses of the DNA and protein sequences revealed that the tos gene encodes a 59,442 Da protein. Extracts containing the overproduced Tos protein markedly increased the formation of ribosomal termination complexes and stimulated the RF-1 or RF-2 activity in the codon-dependent in vitro termination assay. These results fit perfectly with thos … More e described in the original publications on RF-3 and the tos gene has therefore been designated prfC.2. Control and function of lysyl-tRNA synthetases in E. coli : diversity and co-ordination : Lysyl-tRNA synthetases are synthesized from two distinct genes in E.coli, lysS (constitutively) and lysU (inducibly), however, the physiological significance and the differential control mechanism of these two genes have been a long-standing puzzle. We have successfully uncovered a significant control mechanism of lysU expression, that involves the leucine-responsive regulatory protein (Lrp) and a translational enhancer element called "downstream box".3. Regulation of Ty1 transposition and mitochondrial heat shock protein expression by the minor arginine (AGG) tRNA in Saccharomyces cerevisiae : The S. cerevisiae retrotransposon Ty1 genome contains two genes, TYA1 (gag) and TYB1 (pol) genes, and the TYA1-TYB1 fusion protein is synthesized by +1 ribosomal frameshifting in the TYA1 sequence CUU AGG C. Ribosomal pausing at a rare AGG-arginine codon and slippage of a leucyl-tRNA from CUU to UUA are required for frameshifting. Disruption of the tRNA(CCU) gene, designated HSX1, caused increased translational frameshifting and decreased retrotransposition. Disruption of the HSX1 gene also caused a defect in heat-shock induction of the SSC1 gene which encodes a mitochondrial chaperon, hsp70, of S. cerevisiae. Since the hsx1 mutation did not affect expression of other hsp genes, SSC1 expression is likely controlled by a novel mechanism involving the minor tRNA (CCU). Less