Studies in translation discrimination, termination, and frameshifting

This dissertation utilized genetic manipulation of rRNA and mRNA to understand the molecular interactions that are pertinent in translation discrimination, termination and programmed frameshifting.;Discrimination. In Escherichia coli the nucleotides guanosine 530 (G530) and adenosine 1492 (A1492) are located across from each other in the ribosomal A site and play critical roles in the selection of tRNA. Mutations in these positions are dominant lethal independently, but have not been analyzed together. Molecular dynamics simulations predicted that mutations G530 to A and A1492 to G would create ribosomes functionally comparable to wildtype. This work found no mutations in 530 or 1492 that produced significant ribosome activity. Simulations predicted that A1408 may inhibit A1492G mutant activity. Mutations were made in A1408 with 530 and 1492 double mutants. There was no combination of triple mutations created that restored wildtype-comparable activity. It is likely that G530 and A1492 are functionally constrained in ways other than their hydrogen bonding.;Termination. The nucleotide following UGA (UGAN) and mutations in 16S rRNA residue 1054 (normally C1054) can affect stop codon recognition by Escherichia coli release factor 2 (RF2). Their effects on UGA stop codon recognition have not been directly studied together. Analysis of UGAN and 1054 mutation during frameshifting and readthrough events suggested that the two residues do not directly interact to recruit RF2. Two models suggesting the role of both residues in RF2 recruitment are presented.;Frameshift stimulation. EST3 protein is a component of the telomerase holoenzyme in Saccharomyces cerevisiae. The gene requires programmed frameshifting at the sequence CUU-AGU-U to produce a protein required for telomerase activity. The frameshift efficiency of the complete EST3 gene is 45%. However the frameshifting efficiency at the sequence CUU-AGU-U alone is only 6%. These data suggest that since the observed level of frameshifting in the EST3 gene is much higher than the EST3 frameshift sequence alone, the higher level of frameshifting is generated by a contribution from the gene's context. A 27 nucleotide sequence, called the EST3 stimulator, was isolated downstream from the frameshift site that stimulates frameshifting 8 fold.