This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The goal of this project is to understand a unique mechanism of transcription elongation control originally discovered in the bacteriophage HK022. Most members of the lambda phage family use phage-encoded proteins to promote early gene expression by suppressing transcription termination. HK022 does not encode an antitermination protein but relies instead on the direct interaction of sites in the nascent transcript with RNA polymerase. The specific hypothesis is that the activity of RNA-based antiterminators depends upon the recognition of sequence and structural information in the nascent transcript by RNA polymerase. This hypothesis is based upon previous studies that 1) have shown that mutations that disrupt base pairing in the RNA reduce antitermination and secondary mutations that re-establish base pairing restore terminator read through 2) interchanging segments of antiterminator RNAs drastically affects activity, and 3) RNA-mediated antitermination is blocked by mutations in the beta prime subunit of E. coli RNA polymerase. The goal of this proposal is to identify the required sequence and structural elements of RNA based antiterminators.
The specific aims are: 1) to identify additional examples of antiterminator RNAs in lambdoid phages;2) to use in vivo, in vitro and in silico approaches to determine the structurally and functionally important features of the newly identified antiterminator RNAs;and 3) to complete the annotation of two new phage genomes that possess RNA-based antiterminators. The antiterminator sequences discovered in HK022 provide unique examples of RNAs that control gene expression by directly modifying the transcription apparatus. Unusual modes of gene regulation are potential targets for drug design. Therefore, a better understanding of antiterminator RNAs and their recognition by RNA polymerase may facilitate the discovery or development of therapeutic agents capable of altering the expression of virulence genes and thus attenuating disease processes.
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