Date of Award

1992

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Abstract

The synthesis of RNA polymerase in E. coli appears to be limited by the synthesis of the {dollar}\beta{dollar} and {dollar}\beta\sp\prime{dollar} subunits the genes for which (rpoB and rpoC respectively), are the distal components of an operon which also includes the genes for four ribosomal proteins. Studies from several laboratories suggest that RNA polymerase is involved in the regulation of its own synthesis via an autogenous feedback mechanism. The RNA polymerase genes cannot be readily manipulated in vivo in order to study their autogenous regulation since many useful regulatory mutations are lethal. To circumvent these problems transcriptional and translational gene fusions of rpoB to the reporter gene lacZ were constructed on {dollar}\lambda{dollar} phage. To examine feedback regulation the normal synthesis of the {dollar}\beta{dollar} subunit was perturbed by augmenting the production of {dollar}\beta{dollar} and {dollar}\beta\sp\prime{dollar} via an expression plasmid in lysogens of these phages. The results from {dollar}\beta{dollar}-galactosidase assays and immunoprecipitation of the fusion protein argue that in vivo, the synthesis of {dollar}\beta{dollar} is regulated at the translational level by a feedback mechanism.;The various expression plasmids utilized in this study resulted in differing levels of production of the {dollar}\beta{dollar} subunit. Via measurements of individual subunit synthesis, mRNA production and {dollar}\beta{dollar}-galactosidase production from genetic constructs which contain varying amounts of the region upstream or downstream of the start of rpoB, the amount of sequence required for the efficient translation of {dollar}\beta{dollar} subunit was further delineated to a 5{dollar}\sp\prime{dollar} region that starts between 79 and 43 bp upstream of rpoB. Based upon the results of rpoB-lacZ fusions it also appears that more than the first 29 bp of rpoB downstream of the translational initiation site is required for efficient translation.;Measurements of {dollar}\beta{dollar}-galactosidase production after overproduction of {dollar}\beta{dollar} and {dollar}\beta\sp\prime{dollar} in lysogens of rpoB-lacZ translational fusion phage in which varying amounts of the region upstream or downstream of the start of rpoB were utilized argue that the feedback site appears to lie in a region between {dollar}-{dollar}29 bp to +126 bp relative to the start of rpoB.

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