The process of copying information contained in the DNA of an organism into RNA by RNA polymerase is a crucial step in the biological expression of this information. Consequently, this process is an important junction at which control of gene expression can be exerted and as such warrants our continued efforts towards its understanding. RNA polymerase from E. coli has in common with other RNA polymerase the unique ability to cause local strand separation at its specific binding sites (i.e. promoters) on DNA.
The aim of this proposal is to enhance our understanding of the mechanism by which this is accomplished and to be able to describe the effects of conditions that alter promoter utilization by RNA polymerase in terms of this mechanism. Two promoters will be studied. The PRM promoter of bacteriophage lambda plays an important role in the selection of which one of two mutually exclusive developmental pathways will be followed upon infection of a bacterial cell by the phage. The rrn B P1 promoter directs the synthesis of bacterial ribosomal RNA. Its activity is sensitive to the growth rate of the cell. The study of the interplay between two DNA regions contacted by RNA polymerase and the spacer DNA separating them will continue to be a main focus of research. The proposed work is carried out using promoters with particular sequence alterations, constructed in vitro, then cloned. Examples of changes in the spacer DNA include those that alter the length and/or the structure of the DNA. The effect of these alterations on the in vivo and in vitro properties of the promoters will be studied to further probe the mechanisms of functional complex formation, and the determinants of growth rate control. Examples of changes in the spacer DNA that will be studied include those that alter the length and/or the structure of the spacer DNA, and those that result in an altered control of a particular promoter to variations in growth rate. As a model system for studying the activation of functional complex formation by regulatory proteins, the conditions for stimulation of the PRM promoter of phage lambda by the phage-encoded cI protein will be studies in detail, using several of the promoter constructs needed for the studies described above. It is expected that both kinetic and structural factors serve to define a """"""""window"""""""" of conditions that are compatible with the stimulatory effect of the protein.
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