A mutant RNA polymerase reveals a kinetic mechanisms for the switch between nonproductive stuttering synthesis and productive initiation during promoter clearance.

During transcription initiation from galP2, one of the two promoters of the Escherichia coli galactose operon with an initially transcribed sequence of pppAUUUC, RNA polymerase (RNAP) is known to engage nonproductive stuttering synthesis, which is sensitive to the concentration of UTP. This study examines the effect of this nonproductive synthesis on promoter clearance and determines other parameters that might affect stuttering synthesis by analyzing a mutant RNAP, RpoB3449, that has altered its function at this process at galP2. RpoB3449 has dramatically diminished stuttering synthesis, and consequently, it has increased the rate of productive initiation due to its enhanced rate of promoter clearance of galP2 compared with wild-type RNAP. Thus, a direct linkage between promoter clearance and productive transcription is demonstrated. The mechanism by which the mutant RNAP has altered the switch between nonproductive stuttering synthesis and productive initiation during promoter clearance is studied. Apparently, RpoB3449 has increased its efficiency in incorporating CTP at the +5 position of the galP2 transcript leading to its reduced stuttering synthesis, indicating that the rate of an RNAP incorporating the CTP after a stretch of uridine residues is important for promoter clearance at galP2. Because RpoB3449 demonstrates "wild-type" stuttering synthesis at the mutant galP2 promoter, which contains the 6 residue at the +5 position, it indicates that the mutant RNAP has altered in binding CTP at this context. Further experiments indicate that it is the +5 position per se of the galP2 sequence rather than a particular nucleotide at that position that is critical in determining the switch between the two alternate pathways during transcription initiation. A checkpoint model for the switch between nonproductive and productive initiations during promoter clearance is discussed.