A structure/function analysis of the interaction of the Escherichia coli NusA protein with RNA polymerase, the phage lambda N protein, and nut site RNA

Abstract

grantor: University of TorontoNusA is an 'E. coli' protein that controls transcription elongation, termination and antitermination. In this thesis, I show that the functions of NusA in transcription are facilitated by interactions with RNA polymerase, RNA and the [lambda] N protein. Use of a series of deletion constructs of NusA allowed me to identify specific regions of NusA involved in specific interactions and in various aspects of NusA function. Genetic evidence suggested that NusA may interact with the 'boxA ' portion of the N-utilization site ('nut' site= ' boxA, interbox,' and 'boxB'). By constructing multiple nucleotide substitutions in the 'nut' site, I showed that the identities of certain nucleotides at the 3' end of ' boxA' and in the 'interbox ' were important for NusA to associate with an N-'nut' site complex. NusA association with RNA in the presence of N is presumably facilitated by its S1 and KH homology regions, two types of RNA-binding domains in NusA. Elimination or mutation of the S1 homology region prevented the association of NusA with an N-' nut' site complex. Using affinity chromatography experiments, I found that RNA polymerase bound equally well to an amino-terminal RNA polymerase-binding region in amino acids 1-137 and a carboxy-terminal RNA polymerase-binding region in amino acids 232-495 of NusA. By contrast, the à subunit of RNA polymerase only bound to the carboxy-terminal RNA polymerase-binding region of NusA. N protein also bound to a carboxy-terminal region of NusA, and both N and à allowed NusA to associate with RNA in a gel mobility shift assay. When the carboxy-terminal region of NusA was deleted in NusA (1-348), the loss of N-binding and Ã-binding ability did not abolish NusA function in termination and antitermination assays. This minimal functional NusA protein retained the KH and S1 homology regions and the amino-terminal RNA polymerase-binding region. Unlike full length NusA (1-495), NusA (1-416) could bind RNA on its own. These observations suggest that the carboxy-terminal region of NusA inhibits RNA binding and that this inhibition can be relieved by interaction with the [lambda] N protein or the à subunit of RNA polymerase.Ph.D