Characterization of 5'-translational Blockage in E.coli

The 61 sense codons in the universal genetic code encode 20 amino acids, leading to a redundancy such that, for most amino acids, a given amino acid is encoded by multiple, or synonymous, codons. The usage of synonymous codons in protein-coding sequences is highly non-random within any given organism and varies widely from organism to organism. There have been a number of studies in recent years assessing the impact of this codon bias on protein synthesis. It appears that high-usage codons facilitate maximal rates of protein synthesis, while some low-usage codons are inhibitory, at least when they are consecutively placed. In some of the studies on effects of low-usage codons, an unexpected phenomenon was observed: the extent of inhibition of protein synthesis conferred by these low-usage codons was much greater the closer these codons were to the translation start site. Because of the proximity of translation start sites to 5' ends of messages, this phenomenon has been termed '5'-translational blockage'.

This project is concerned with characterization of 5'-translational blockage in E. coli. The working hypothesis is that 5'-translational blockage is a result of ribosomes and/or the translational apparatus behaving differently during elongation near translation starts compared to downstream. To begin the process of determining the basis of this phenomenon, the investigators will develop second-generation codon usage vectors that encode mRNA with reduced stability, with the prediction that such vectors will exacerbate the phenomenon (thereby making it easier to study). This prediction is based on theoretical considerations; however, even if the prediction does not prove true, the question of effects, if any, of varying mRNA stability is of intrinsic interest. The implication of 5'-translational blockage is that ribosomes and/or the translational apparatus are functionally different near translation starts compared to positions downstream on the message. Understanding this surprising and exciting phenomenon will not only further our knowledge of the mechanism of protein synthesis, but may also have important implications for bioengineering expression of heterologous genes.