GreA and GreB proteins revive backtracked RNA polymerase in vivo by promoting transcript trimming.
The GreA and GreB proteins of Escherichia coli show a multitude of effects on transcription elongation in vitro, yet their physiological functions are poorly understood. Here, we investigated whether and how these factors influence lateral oscillations of RNA polymerase (RNAP) in vivo, observed at a protein readblock. When RNAP is stalled within an (ATC/TAG)(n) sequence, it appears to oscillate between an upstream and a downstream position on the template, 3 bp apart, with concomitant trimming of the transcript 3' terminus and its re-synthesis. Using a set of mutant E.coli strains, we show that the presence of GreA or GreB in the cell is essential to induce this trimming. We show further that in contrast to a ternary complex that is stabilized at the downstream position, the oscillating complex relies heavily on the GreA/GreB-induced 'cleavage-and-restart' process to become catalytically competent. Clearly, by promoting transcript shortening and re-alignment of the catalytic register, the Gre factors function in vivo to rescue RNAP from being arrested at template positions where the lateral stability of the ternary complex is impaired.
Toulmé
F
F
Centre de Biophysique Moléculaire, CNRS, rue Charles Sadron, 45071 Orléans cédex 2, France.
Mosrin-Huaman
C
C
Sparkowski
J
J
Das
A
A
Leng
M
M
Rahmouni
A R
AR
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D007700:Kinetics; D009154:Mutation; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C079667:GreB protein, E coli; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C075886:GREA protein, Rickettsia prowazekii; D012321:DNA-Directed RNA Polymerases