Novel aspects oftrp repressor function
Abstract
The trpR gene of Escherichia coli encodes a polypeptide of 108 amino acids. In dimeric form, the TrpR protein transcriptionally regulates several key enzymes of aromatic amino acid biosynthesis and transport. The nucleotide sequence of the Salmonella typhimurium trpR gene (also 108 codons) was determined based on homology to the E. coli chromosomal trpR sector by using polymerase chain reaction (PCR) methodologies. The inferred amino acid sequence of the S. typhimurium protein differs at three positions from its E. coli counterpart. A large collection of trpR mutants was characterized by sequencing PCR amplified DNA. This approach minimized the possibility of in vivo selection against mutant trpR genes. Altogether, 22 previously undescribed amino acid switches that impose a TrpR phenotype were identified. There are now 43 locations within the TrpR protein where a loss of DNA-binding function accompanies an amino acid switch. In three independent cases, TrpR$\sp-$ phenotypes resulted from changes at the TGA stop codon of the trpR open reading frame (ORF). The creation of sense codons, at the stop codon of trpR, extended the trpR ORF by 50 codons and incorporated a Repetitive Extragenic Palindrome (REP element) into a new chimeric gene. The incorporation of a REP element into the trpR ORF abolishes repressor function owing to the inability of the translational machinery to cope with an unusual number of hungry codons (rare codons with low cytoplasmic levels of their cognate tRNA). Such codons are an integral feature of REP elements and may dictate that these elements be extragenic. The availability of the entire genomic sequence of Haemophilus influenzae prompted a structure-function analysis of the TrpR protein of this organism. The H. influenzae TrpR protein is inferior to that of E. coli in its ability to repress promoters of the E. coli TrpR regulon. The overall regulation of the cytoplasmic levels of the H. influenzae TrpR protein differs from that of E. coli, in that proteolysis appears to be an important factor in dictating cytoplasmic levels. The two evolutionarily distinct TrpR monomers do appear to interact to form heterodimers.
Degree
Ph.D.
Advisors
Somerville, Purdue University.
Subject Area
Molecular biology|Genetics|Biochemistry
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