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Effect of low nucleotide concentrations on abortive elongation catalysed by wheat-germ RNA polymerase II.

ABSTRACT: A kinetic study of the effect of elongating nucleotide concentration on the reactions of abortive elongation catalysed by wheat-germ RNA polymerase II on a poly[d(A-T)] template suggests that the shift from abortive to productive elongation may involve the participation of at least two nucleotides, according to a mechanism very similar to that reported for Escherichia coli RNA polymerase. Experiments performed with non-complementary nucleotides with respect to the DNA template, and with substrate derivatives, allow an analysis of the substrate specificity during these reactions. Similar experiments performed with poly[d(A-A-T)].poly[d(T-T-A)] as template provide a starting point for a better understanding of the effect of DNA sequence on the rates of abortive and productive elongation catalysed by the plant enzyme.

SUBMITTER: Job C

PROVIDER: S-EPMC1147966 | biostudies-other | 1987 May

REPOSITORIES: biostudies-other

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Project description:Abortive cycling is a universal feature of transcription initiation catalyzed by DNA-dependent RNA polymerases (RNAP). In bacteriophage T7 RNAP, mutation of proline 266 to leucine (P266L) in the C-linker region connecting the N-terminal promoter binding domain with the C-terminal catalytic domain drastically reduces short abortive products (4-7 nt) while marginally increasing long abortives (9-11 nt). Here we have investigated the transcription initiation pathway of P266L with the goal of understanding the mechanistic basis for short and long abortive synthesis. We show that the P266L mutation does not alter the affinity for the promoter, mildly affects promoter opening, and increases the +1/+2 GTP K(d) by 2-fold. However, unlike wild-type T7 RNAP that undergoes stepwise rotation of the promoter binding domain and DNA scrunching during initial transcription, the P266L mutant does not undergo coupled rotational/scrunching movements until 7 nt RNA synthesis. The lack of rotation/scrunching correlates with greater stabilities of the initiation complexes of the P266L and decreased short abortive products. The results indicate that the increased flexibility in the C-linker due to P266L mutation enables T7 RNAP to absorb the stress from the growing RNA:DNA hybrid thereby decreasing short abortive products. Increased C-linker flexibility, however, has an adverse effect of delaying the transition into elongation by 1-2 nt, which gives rise to long abortive products. However, a mutation in the upstream promoter region greatly decreases long abortive products in P266L reactions, rendering the combination of P266L and A-15C promoter a desirable pair for efficient in vitro transcription for RNA production. We conclude that the conformational rigidity in the C-linker region conferred by the proline at position 266 is responsible for the undesirable short abortive products, but the rigidity is critical for efficient promoter clearance and transition into elongation.

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Effect of low nucleotide concentrations on abortive elongation catalysed by wheat-germ RNA polymerase II.

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