Structural and functional studies of the Escherichia coli cyclic AMP receptor protein.
Item
-
Title
-
Structural and functional studies of the Escherichia coli cyclic AMP receptor protein.
-
Identifier
-
AAI9521283
-
identifier
-
9521283
-
Creator
-
Jin, Ruzhong.
-
Contributor
-
Adviser: Joseph S. Krakow
-
Date
-
1995
-
Language
-
English
-
Publisher
-
City University of New York.
-
Subject
-
Biology, Microbiology | Biology, Molecular | Biology, Cell
-
Abstract
-
The Escherichia coli cAMP protein, which is referred to as CRP or CAP, is an allosteric transcriptional regulatory protein which regulates the transcription of more than 20 genes in E. coli. The relationship between CRP structure and how it mediates positive control of gene expression is investigated in this dissertation. One surface exposed loop of CRP located at amino acids 156-162 has been shown to be involved in contact with the {dollar}\alpha{dollar} subunit of RNA polymerase and important in activating transcription. Insertion mutants were generated in this loop. All insertion mutants retained normal DNA binding activity and showed fairly high activity on the gal P1 (class II promoter, centered at {dollar}-{dollar}41.5) compared to wild type CRP. But several showed defective positive control activity on the lac P1 (class I promoter, centered at {dollar}-{dollar}61.5). The results indicate that the conformation of the 156-162 loop of CRP is important in determining a functional interaction with RNA polymerase. Another surface exposed loop at around amino acid 52 is also considered to be a potential candidate for transcription activation. Using protein-protein photocrosslinking, the results showed that the 52-loop of CRP is in physical proximity to the {dollar}\sigma{dollar} subunit of RNA polymerase. The superactive CRP suppressor mutant, K52N, increased the efficiency of crosslinking, indicating that the K52N mutation improved the physical interaction between the CRP 52-loop and the {dollar}\sigma{dollar} subunit. As a control, the 156-162 loop was found to crosslink only with the RNA polymerase {dollar}\alpha{dollar} subunit both on lac and gal promoters in the presence of cAMP, indicating that the 156-162 loop is close to the {dollar}\alpha{dollar} subunit on the gal promoter as well as on the lac promoter.;Five conserved glycine residues at positions 33, 45, 56, 67 and 71 of CRP were substituted with alanine; G33C, G56C and G67C showed comparable in vivo activity on both lac and gal promoters with the wild type CRP. G71C showed only {dollar}{lcub}\sim60{rcub}{dollar}% of the wild type activity and G45C was almost totally inactive. Western blotting indicated that G45C is a very unstable protein in vivo which may explain its low activity.
-
Type
-
dissertation
-
Source
-
PQT Legacy CUNY.xlsx
-
degree
-
Ph.D.
