The glutamic acid residue at amino acid 261 of the alpha subunit is a determinant of the intrinsic efficiency of RNA polymerase at the metE core promoter in Escherichia coli.
A mutation in the rpoA gene (which encodes the alpha subunit of RNA polymerase) that changed the glutamic acid codon at position 261 to a lysine codon decreased the level of expression of a metE-lacZ fusion 10-fold; this decrease was independent of the MetR-mediated activation of metE-lacZ. Glutamine and alanine substitutions at this position are also metE-lacZ down mutations, suggesting that the glutamic acid residue at position 261 is essential for metE expression. In vitro transcription assays with RNA polymerase carrying the lysine residue at codon 261 indicated that the decreased level of metE-lacZ expression was not due to a failure of the mutant polymerase to respond to any other trans-acting factors, and a deletion analysis using a lambda metE-lacZ gene fusion suggested that there is no specific cis-acting sequence upstream of the -35 region of the metE promoter that interacts with the alpha subunit. Our data indicate that the glutamic acid at position 261 in the alpha subunit of RNA polymerase influences the intrinsic ability of the enzyme to transcribe the metE core promoter.
Jafri
S
S
Department of Microbiology, University of Iowa, Iowa City 52242, USA.
Urbanowski
M L
ML
Stauffer
G V
GV
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D000409:Alanine / Q000737:chemistry; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003062:Codon; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D018698:Glutamic Acid / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D008780:Methyltransferases; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D013489:Suppression, Genetic; D015534:Trans-Activators / Q000235:genetics; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D003062:Codon; D029968:Escherichia coli Proteins; C058169:MetR protein, Bacteria; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D018698:Glutamic Acid; D008780:Methyltransferases; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; C058170:metE protein, E coli; D012321:DNA-Directed RNA Polymerases; D008239:Lysine; D000409:Alanine