Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2011.
The ribosome decodes mRNA messages and catalyzes protein
synthesis (a process termed translation), making it one of the most
fundamental structures in any living cell. Biochemical studies over the past
60 years and structural data produced over the last decade have revealed the
mechanism of translation in remarkable detail. In contrast, ribosome
biogenesis, the process by which the ribosome is produced in vivo, remains
largely enigmatic; this is due in part to the redundancy of processes involved,
its efficient nature, and the multi-faceted nature of many extra-ribosomal
biogenesis factors. Work presented in this thesis uses genetic and
biochemical tools to overcome these obstacles and define the role for KsgA, a
universally conserved small ribosomal subunit (SSU) rRNA modification
enzyme, in facilitating a checkpoint in the SSU biogenesis program. Indeed,
genetic removal of the KsgA checkpoint and concomitant overexpression of
RbfA, an incompletely characterized ribosome biogenesis factor, results in
incorporation of immature SSUs in 70S ribosomes, and an accumulation of
intermediates of SSU biogenesis. Moreover, the methylation activity of KsgA
is a vital component of this checkpoint function as overexpression of
methyltransferase deficient KsgA impedes SSU biogenesis and results in an
accumulation of an intermediate of the SSU biogenesis program.
Biochemical analysis of SSU intermediates and aberrant 70S ribosomes that
accumulate under these conditions has provided chronological details of SSU
biogenesis events and an unprecedented look at the transition of the SSU
from the biogenesis program to the translation cycle. Work herein describes,
for the first time, a checkpoint role for KsgA, a universally conserved
methyltransferase, and a switch function for the methylations. In addition,
interactions between KsgA and other biogenesis factors has revealed details
of interplay between ribosome biogenesis and translation initiation.
