Escherichia coli RNA polymerase mutations located near the upstream edge of an RNA:DNA hybrid and the beginning of the RNA-exit channel are defective for transcription antitermination by the N protein from lambdoid phage H-19B.
Transcription antitermination is an important mechanism that can control regulation of gene expression. The N protein of lambdoid phages modifies the transcription elongation complex (EC) and helps it to overcome downstream terminators. In this modified EC, the C-terminal domain of N makes specific interactions with RNA polymerase (RNAP). The interacting surface of RNAP for N is unknown. Here, we report five mutations in the beta (G1045D) and beta' (P251S, P254L, R270C and G336S) subunits of RNAP that are specifically defective for antitermination by N protein of the lambdoid phage, H-19B. A mutation in the C-terminal domain of N, L108F, suppresses the defect of beta'-P254L. Purified mutant holoenzymes exhibit less processive antitermination. The amino acid substitutions in the mutant RNAPs cluster very close to the RNA:DNA hybrid at the beginning of the RNA-exit channel of the EC. We suggest that the action of H-19B N is exerted through the region defined by these amino acids. Wild-type N stabilizes the EC at terminator sites and in this modified EC a part of the terminator hairpin may form but appears to be unstable. We propose that the action of N close to the active center alters the RNAP-nucleic acid interactions around the RNA:DNA hybrid, which impairs proper folding of the terminator hairpin or stabilizes the weak RNA:DNA hybrid, or both.
Cheeran
Anoop
A
Laboratory of Transcription Biology, Center for DNA Fingerprinting and Diagnostics, Nacharam, Hyderabad-500076, India.
Babu Suganthan
Rajan
R
Swapna
G
G
Bandey
Irfan
I
Achary
M Sridhar
MS
Nagarajaram
H A
HA
Sen
Ranjan
R
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic; D017361:Viral Nonstructural Proteins
D029968:Escherichia coli Proteins; D017361:Viral Nonstructural Proteins; D012321:DNA-Directed RNA Polymerases