The translational bypassing in the Phage T4 gene 60 represents the efficient synthesis of one protein from two coding sequences that are separated by 50 nucleotides in mature mRNA. This example challenges our knowledge of protein synthesis and our confidence in predictions of the variety of protein products encoded in genomes. The overall aim of the proposed work is to understand the mechanisms involved in the best-studied case of translational discontiguity. This will be achieved by elucidating why there is a preference for coding resumption after 50 nucleotides. Up-mutants will be isolated and characterize that give efficient bypassing from cassettes with different defects in the gene 60 bypassing signals: poorly functioning matched take-off and landing codon pairs, extended coding gap, extended stem loop and related studies with mutants of nascent peptide affecting frame shifting. Starting with a WT gene 60 bypassing cassette, to isolate down-mutants with screening rather than selection, and repeat in a strain with a single copy of ribosomal RNA genes. To gain functional information from genetic studies about the interacting partners of ribosomal protein L9 that is important for bypassing. To facilitate cryo-electron microscopic studies by a collaborator of bypassing and frameshifting to gain structural information. To investigate pausing in bypassing and frameshifting. Recoding: Redefinition and Frameshifting Reprogramming of the readout of certain mRNAs can cause a different meaning for a code word or alternative reading frames to be accessed. Here, dynamic redefinition of the """"""""stop"""""""" codon of Murine Leukemia Virus (MuLV) gag gene and the ribosomal frame-shifting required for expression of mammalian antizyme are to be studied. This frame-shifting serves as the sensor of an autoregulatory circuit. For both redefinition and frame-shifting, signals in mRNA stimulate the efficiency of the non-standard event at the recoding site. To ascertain if possible structural elements that include the recoding site exist and if so whether they play a synergistic role in antizyme frame shifting and MuLV gag-pol stop codon read through. To determine if there is tissue specificity to the programmed frame-shifting candidate, antizyme 3, by using a transgenic mouse approach. Recoding: Further mechanistic studies partly as a guide for identifying new cases To determine the effects of mutations of E. coli tRNA Gly2 , isolated for their bypassing and frame-shifting properties, on tRNA structure and dynamics. To determine the solution structure of E.coli tRNA Ala GCC because of the evidence for the purine at position 32 influencing frame-shifting. To investigate a role for the base at tRNA position 34 or 37 on E. coli-1 frame-shifting: single tRNA slips at a hexa-nucleotide shift site in distinction to the classical tandem shifts at hepta-nucleotide sites. To keep monitoring newly reported sequences for potential new cases and to respond to suggestions of others who have encountered new cases.
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