The major product of cellular transcription is ribosomal RNA, which can make up 80-90 percent of the total RNA in the cells of prokaryotes and eukaryotes. The long-range goal of this study is to understand how ribosomal RNA (rrn) gene expression is regulated, both at the genetic and biochemical levels, in response to changes in environmental conditions. We will continue by addressing the following aspects of rrn expression: 1. The role of antitermination in rrn expression. The boxB-BoxA antiterminator (AT) motif in rrn leader and spacer regions can be traced through most phyla in the eubacterial and archaebacterial kingdoms. Occurrence of this pattern in other bacteria as well as E. coli suggests that rrn antitermination mechanisms are wide spread in nature and have been conserved in microbial evolution. The unique arrangement of AT sequences in E. coli rrn leader and spacer regions supports the idea that the antitermination mechanism may have more than one function; in addition to suppression of transcription termination, AT sequences may also facilitate rRNA processing and may insure that the RNA is folded properly. We will use two approaches to address this topic. The first will employ electron microscopy to directly examine how RNA polymerase molecules transcribe an rrn transcription unit that has a mutated AT sequence, and to visualize whether processing occurs in an AT mutant operon. In the second approach we will place """"""""reporter"""""""" segments within the rrn operon spacer regions and monitor their transcription as influenced by wild type and mutant AT sequences. 2. Isolation and characterization of antiterminator suppressor mutations. We have isolated a large number of rrn AT mutations which have lost terminator read-through ability. These mutations will allow us to isolate second site mutations which suppress the mutant AT phenotype. These suppressors will be used to probe the mechanism of rrn antitermination. We will combine mutagenesis with temperature selection to provide the widest possible range of suppressor selections. 3. Antitermination of promoters that use other sigma factors. Initial experiments combining the rrn AT with promoters that use different sigma factors gave two unexpected results. Transcripts were not antiterminated, yet the presence of the AT increased expression by 10-fold. Using operon fusion plasmids that we have constructed, we will alter the spacing between the promoter and the AT, and will use mutations and subfragments of the AT to determine how they influence our previous results. 4. Terminators and rrn antitermination. We plan to define the features that are required for efficient and for dissociation of the RNA polymerase antitermination complex. To do this, we will dissect several rrn terminators using a combination of DNA synthesis, BAL31 deletions, and mutagenesis. 5. Regulatory studies of rrn expression. We will examine several specific features of rrn regulation. We will recombinant technology and genetics to examine the comparative expression of the redundant operons, the influence of regions 5' of the operons on their expressions, and the effect of rrn operon inactivation on cell growth and viability. we will use in vitro biochemical techniques to examine antitermination in a purified in vitro transcription system.
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