| Research Abstract |
The DNA-dependent RNA polymerase of Escherichia coli is composed of four major subunits, alpha, beta, beta', and sigma, and carries not only the catalytic activity of RNA synthesis but also complex functions of transmission of genetic information. In order to understand the structural bases for these multiple functions, we carried out a systematic analysis of functional domains on each subunit of E. coli RNA polymerase and obtained the following results.
1) The alpha subunit plays an essential role in the assembly of two large subunits, beta and beta', into enzyme complex. The N-terminal region is required for the sub unit assembly, while the C-terminal domain is needed for transcription activation by position factors which bind DNA upstream of the promoter -35 signal. Mutant RNA polymerases consisting of C-terminal truncated alpha subunits are , however, activated by positive factors which bind to target sites that overlap the promoter -35 region.
2) Four functional sites have been mapped on the beta subunit, including rifampicin bindint site, ppGpp sensitivity control site, promoter selection site and sigma binding site.
3) Putative novel sigma subunits have been identified, using antibodies against synthetic peptides with consensus amino acid sequences common among various sigma subunits. Genes coding for these proteins are being cloned using DNA probes designed based on these amino acid sequences.
4) The functions of these basic subunits are modulated by interaction with accessory transcription factors or modification of enzyme subunits. Sub unit omega is needed for interaction with ppGpp, a regulatory molecule in stringent control.
5) Novel modified forms of RNA polymerase holoenzyme exist in stationary-phase E. coli cells, each recognizing a different set of promoters.
These results altogether indicate that global control of gene transcription takes place mainly by modulating the functions of RNA polymerase.
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