Thermal probing of E. coli RNA polymerase off-pathway mechanisms.
RNA polymerase (RNAP) is an essential enzyme for cellular gene expression. In an effort to further understand the enzyme's importance in the cell's response to temperature, we have probed the kinetic mechanism of Escherichia coli RNAP by studying the force-velocity behavior of individual RNAP complexes at temperatures between 7 and 45 degrees C using optical tweezers. Within this temperature range and at saturating nucleotide concentrations, the pause-free transcription velocity of RNAP was independent of force and increased monotonically with temperature with an elongation activation energy of 9.7+/-0.7 kcal/mol. Interestingly, the pause density at cold temperatures (7 to 21 degrees C) was five times higher than that measured above room temperature. A simple kinetic model revealed a value of 1.29+/-0.05 kcal/mol for the activation energy of pause entry, suggesting that pause entry is indeed a thermally accessible process. The dwell time distribution of all observable pauses was independent of temperature, directly confirming a prediction of the model recently proposed for Pol II in which pauses are diffusive backtracks along the DNA. Additionally, we find that the force at which the polymerase arrests (the arrest force) presents a maximum at 21 degrees C, an unexpected result as this is not the optimum temperature for bacterial growth. This observation suggests that arrest could play a regulatory role in vivo, possibly through interactions with specific elongation factors.
Mejia
Yara X
YX
Graduate Group in Applied Science and Technology, University of California, Berkeley, CA 94720, USA.
Mao
Hanbin
H
Forde
Nancy R
NR
Bustamante
Carlos
C
D004269:DNA, Bacterial / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D052898:Optical Tweezers; D012329:RNA, Bacterial / Q000737:chemistry; D013314:Stress, Mechanical; D013696:Temperature; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012321:DNA-Directed RNA Polymerases