Elucidating Ribosomes-Genetic Studies of the ATPase Uup and the Ribosomal Protein L1.

The ribosome is comprised of two subunits, formed from three ribosomal RNAs and many ribosomal proteins. The two subunits come together around a messenger RNA during initiation, and catalyze the addition of sequential amino acids to a growing polypeptide chain as directed by the messenger RNA during elongation. While much of the ribosome is a relatively rigid molecular machine, there are two flexible protrusions comprised of rRNA and ribosomal proteins. One of these protrusions is the L1 arm where deacylated tRNAs leave the ribosome. The contributions of the L1 stalk and associating factors are unclear. In this work, I report that loss of the protein L1 results in changes in translation accuracy and perturbation of the ribosome profile. I also find that proteins involved in RpoS regulation/regulon, proteins affecting the structure of the ribosome in the vicinity of the active site, and proteins affecting DNA replication are able to suppress, when at high copy, the previously-­‐reported slow growth the of ΔrplA (encoding L1) mutant. Recent studies have revealed that translation factors act on the L1 arm side of the ribosome. I have characterized Uup, an E. coli ABCF protein that shares many of the residues involved in binding to tRNAfMet and ribosomal protein L1 in the closely related E. coli ABCF protein EttA. Exogenous Uup suppresses many phenotypes caused by deletion of tthe elongation family GTPase BipA, including a growth-­dependent ribosome profile defect. Both translation of reporter plasmids and translating ribosomes are reduced in Δuup. While Δuup and ΔettA show different phenotypes in multiple assays, each can suppress defects resulting from loss of the other. I propose that Uup, as shown for EttA, can promote formation of the first peptide bond in the elongation phase of translation. This adds to the short but rapidly growing list of translation factors regulating translation at the E­‐site.PhDMolecular, Cellular and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/116786/1/katharyn_1.pd