Genetic analysis of RNA polymerase/transcriptional activator interactions at thepepT promoter of S. typhimurium
Lombardo, Mary-Jane
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https://hdl.handle.net/2142/22262
Description
Title
Genetic analysis of RNA polymerase/transcriptional activator interactions at thepepT promoter of S. typhimurium
Author(s)
Lombardo, Mary-Jane
Issue Date
1995
Doctoral Committee Chair(s)
Miller, Charles G.
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Genetics
Biology, Microbiology
Language
eng
Abstract
The role of the alpha subunit of RNA polymerase (encoded by rpoA) in transcriptional activation by the global regulatory protein FNR has been investigated using a genetic approach. We have identified five rpoA mutations which decrease the FNR-dependent anaerobic induction of the pepT gene of S. typhimurium. Each of these mutations (G311E, G311R, L289F, R317H, W320ter) affects an amino acid in the C-terminal region of the alpha subunit. Phenotypic characterization shows that four of the five mutations have relatively specific effects on positively regulated transcription of the pepT gene and other FNR-dependent genes. We propose that the C-terminal region of the alpha subunit is involved in direct interactions with FNR. We reasoned that suppressor analysis might be a useful approach to identifying regions of FNR that are involved in interactions with RNA polymerase. Reversion of the G311E and G311R mutations led to the isolation of six mutations (E47K, T80I, A172V, A172T, V208A, and V208G) in the transcriptional activator FNR which restore or partially restore anaerobic induction of pepT in the presence of each of the five rpoA mutations. A172 and V208 both lie in the DNA binding domain of FNR suggesting that the mutations at these positions might restore activation by affecting DNA binding directly or indirectly. The T80I and E47K mutations lie elsewhere in the protein and are unlikely to affect DNA binding. We suggest that the T80 and E47 regions of FNR might be involved in direct interactions between FNR and the alpha subunit or another subunit of RNA polymerase. In addition, we have characterized the pepT promoter region. Mutational studies and primer extension analysis demonstrate that pepT is transcribed from two promoters, an FNR-dependent, anaerobically induced promoter (P1), and a constitutive promoter (P2) conferring low level expression. A mutant P1 promoter containing a consensus-10 sequence was shown to be regulated by CRP-cAMP in addition to FNR. The potABCD operon was shown to be divergently transcribed from pepT, and the potABCD promoter was identified.
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