The objectives of this proposal are to identify cellular components involved in ribosomal selection of the translation initiation codon in the yeast Saccharomyces cerevisiae. A combination of the powerful techniques of classical yeast genetics and modern molecular biology will be used to characterize revertants of a previously defined mutant (cycl-362 allele) known to be defective in initiation of translation of CYC1 messenger RNA (Stiles et al., Cell 25:277 (1981)).
Specific aims i nclude the following: (i) isolate revertants of cycl-362 by established selection procedures; (ii) genetically distinguish local (CYC1-linked) from extragenic suppressors; (iii) characterize the CYC1 locus from each by DNA sequence analysis and 5' endpoint transcript mapping; (iv) clone the most appropriate extragenic suppressor of the cycl-362 defect; (v) extensively characterize the cloned suppressor; and (vi) ultimately identify the suppressor-encoded trans-acting factor. Extensive biochemical studies of the translational apparatus has identified the components involved in translation initiation and elongation. However, these results do not account for potential control of gene expression at the translational level. It is clear, however, that a number of genes, including many involved in physiological and developmental control mechanisms, are translationally regulated. In yeast, for example, a nuclear gene involved in coordinate control of amino acid biosynthetic genes is translationally controlled. In higher eukaryotes, genes involved in the heat shock response, light-induced developmental signals, and other processes are translationally regulated. Moreover, recent evidence suggests that the tat gene product from human immunodeficiency virus controls translation of the viral encoded messenger RNAs. Translational control is clearly a significant mechanism for controlling the expression of certain genes, yet our present understanding of translation initiation is insufficient to account for such mechanisms. The research program described herein is designed to address this problem by specifically characterizing the factors involved in selection of the translation initiation codon.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29GM039484-04
Application #
3466786
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1988-08-01
Project End
1993-07-31
Budget Start
1991-08-01
Budget End
1992-07-31
Support Year
4
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Louisiana State University Hsc Shreveport
Department
Type
Schools of Medicine
DUNS #
City
Shreveport
State
LA
Country
United States
Zip Code
71103
Singh, Badri Nath; Hampsey, Michael (2014) Detection of short-range chromatin interactions by chromosome conformation capture (3C) in yeast. Methods Mol Biol 1205:209-18
Goel, Shivani; Krishnamurthy, Shankarling; Hampsey, Michael (2012) Mechanism of start site selection by RNA polymerase II: interplay between TFIIB and Ssl2/XPB helicase subunit of TFIIH. J Biol Chem 287:557-67
Hampsey, Michael; Singh, Badri Nath; Ansari, Athar et al. (2011) Control of eukaryotic gene expression: gene loops and transcriptional memory. Adv Enzyme Regul 51:118-25
Seibold, Steve A; Singh, Badri Nath; Zhang, Chunfen et al. (2010) Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase. Biochim Biophys Acta 1799:575-87
Laine, Jean-Philippe; Singh, Badri Nath; Krishnamurthy, Shankarling et al. (2009) A physiological role for gene loops in yeast. Genes Dev 23:2604-9
Singh, Badri Nath; Ansari, Athar; Hampsey, Michael (2009) Detection of gene loops by 3C in yeast. Methods 48:361-7
Ghazy, Mohamed A; He, Xiaoyuan; Singh, Badri Nath et al. (2009) The essential N terminus of the Pta1 scaffold protein is required for snoRNA transcription termination and Ssu72 function but is dispensable for pre-mRNA 3'-end processing. Mol Cell Biol 29:2296-307