The role of steric constraints vs sequence preference in start site selection by T7 RNA polymerase was investigated by using a series of synthetic promoters in which the preferred template strand 'CC' initiation sequence was moved away from its normal position relative to the -17 to -6 element of the T7 promoter. It was found that the CC sequence directs efficient initiation if placed 1 or 2 nt downstream of its normal position, but not if placed upstream, or more than 2 nt downstream, of +1. Mutagenesis revealed that part of the bias to initiate with GTP is due to an interaction between histidine 784 and the 2-amino group of a guanosine bound in the initiating triphosphate position. This interaction is also important for holding short transcripts within the transcription complex during initial transcription.
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