Regulation of transcription initiation is the major determining event employed by the cell to control gene expression and subsequent cellular processes. The weak promoters, with low basal transcription activities, are activated by activators. Bacteriophage Mu mom gene, which encodes a unique DNA modification function, is detrimental to cell when expressed early or in large quantities. Mu has designed a complex, well-controlled and orchestrated regulatory network for mom expression to ensure its synthesis only in late lytic cycle. The phage encoded transcription activator protein C activates the gene by promoter unwinding of the DNA and thereby recruiting of RNAP to the promoter.
C protein functions as a dimer for DNA binding and transcription activation. Mutagenesis and chemical crosslinking studies revealed that the leucine zipper motif, and not the coiled coil motif in the N terminal region, is responsible for C dimerization. The DNA binding domain of C is a HTH domain which is preceded by the leucine zipper motif. The C protein is one of the few examples in the bacterial proteins containing both leucine zipper and HTH domain.
Most of the transcription activators either influence initial binding of RNAP or conversion of closed to open complex formation. Very few activators act at subsequent steps of promoter-polymerase interaction. Earlier studies showed high level of transcription from a mutant mom promoter, tin7. Addition of C further increased transcription from Ptin7 indicating that C may have a role beyond polymerase recruitment. Each steps of transcription initiation have been dissected using the Ptin7 and a positive control (pc) mutant of C, R105D. The results revealed multi-step transcription activation mechanism for C protein at Pmom. C recruits RNAP at Pmom and subsequently increases the productive RNAP-promoter complex and enhances promoter clearance.
To further understand the C mediated transactivation mechanism, interaction between C and RNAP was assessed. C interacts with holo and core RNAP only in presence of DNA. Positive control mutants of C, F95A and R015D, were found to be compromised in RNAP interactions. These mutants were efficient in RNAP recruitment to Pmom but do not enhance promoter clearance. Trypsin cleavage protection experiment indicated that probably C protein interacts with b¢ subunit of RNAP. Interaction between C and RNAP appears to enhance the formation of productive RNAP-promoter complex leading to promoter clearance.
The connection between activator-polymerase interaction and transcription activation is well documented where the recruitment of RNAP is influenced. In case of activators acting at post recruitment steps of initiation, the role of polymerase contact is poorly understood. Our study shows that activator-polymerase interaction can lead to increased promoter clearance at Pmom by overcoming abortive initiation.
| Identifer | oai:union.ndltd.org:IISc/oai:etd.ncsi.iisc.ernet.in:2005/275 |
| Date | 05 1900 |
| Creators | Chakraborty, Atanu |
| Contributors | Nagaraja, V |
| Publisher | Indian Institute of Science |
| Source Sets | India Institute of Science |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Rights | I grant Indian Institute of Science the right to archive and to make available my thesis or dissertation in whole or in part in all forms of media, now hereafter known. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. |
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