Isolation and structural analysis of the Escherichia coli trp leader paused transcription complex.
Transcription pausing is a key step in many prokaryotic transcription attenuation mechanisms. Pausing is thought to occur when an RNA hairpin forms near the 3' end of a growing transcript. We report here the isolation of the trp leader paused transcription complex containing a defined 92-nucleotide nascent transcript. Digestion of isolated paused complexes with RNase T1 suggests that the trp leader RNA hairpin designated 1:2 forms in the paused transcription complex. The transcription factor NusA alters the RNase T1 digestion pattern of the 92-nucleotide pause transcript in the complex but not the cleavage patterns of purified pause RNA, suggesting that NusA specifically affects the 1:2 hairpin in the paused transcription complex. The isolated paused transcription complex retains the ability to resume transcription. Kinetic studies on the resumption of elongation suggest that NusA is a non-competitive inhibitor of paused complex release and that the Ks for GTP is around 300 microM. RNA polymerase in the paused transcription complex protects approximately 30 base-pairs on both DNA strands from exonuclease digestion.
D001426:Bacterial Proteins; D001483:Base Sequence; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics*; D012329:RNA, Bacterial; D014158:Transcription, Genetic; D014364:Tryptophan / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D012329:RNA, Bacterial; D014364:Tryptophan