No abstract

Like multisubunit RNA polymerases (RNAPs), T7 RNAP frequently releases its transcript over the initial 8 -12 transcribed nucleotides, when it still contacts the promoter. This abortive cycling, which is most prominent with initial sequences that deviate from those of T7 late genes, eventually compromises productive transcription. Starting from an in vivo situation where transcription of a target gene by T7 RNAP is virtually abolished because of extensive abortive cycling, we have selected a mutation in RNAP that restores target gene expression. In vitro, this mutation (P266L) weakens promoter binding but markedly reduces abortive cycling over a variety of initial sequences by stabilizing the transcription complex at nucleotides 5-8. Other substitutions of P266 have similar effects. X-ray data show that during the transition from initial to elongation complex, the N-terminal region undergoes a major structural switch of which P266 constitutes one of the hinges. How the mutation might facilitate this switch is tentatively discussed. On the practical side, the mutation can significantly improve in vitro transcription, particularly from templates carrying unfavorable initial sequences.abortive transcription ͉ in vitro RNA synthesis

RNA polymerases carry out the synthesis of an RNA copy from a DNA template. They move along DNA, incorporate nucleotide triphosphate (NTP) at the end of the growing RNA chain, and consume chemical energy. In a single-molecule assay using the T7 RNA polymerase, we study how a mechanical force opposing the forward motion of the enzyme along DNA affects the translocation rate. We also study the influence of nucleotide and magnesium concentration on this process. The experiment shows that the opposing mechanical force is a competitive inhibitor of nucleotide binding. Also, the single-molecule data suggest that magnesium ions are involved in a step that does not depend on the external load force. These kinetic results associated with known biochemical and mutagenic data, along with the static information obtained from crystallographic structures, shape a very coherent view of the catalytic cycle of the enzyme: translocation does not take place upon NTP binding nor upon NTP cleavage, but rather occurs after PPi release and before the next nucleotide binding event. Furthermore, the energetic bias associated with the forward motion of the enzyme is close to kT and represents only a small fraction of the free energy of nucleotide incorporation and pyrophosphate hydrolysis.