We have analyzed the elongation properties of @entity6621 RNA polymerase during a single round of transcription in vitro. RNA-labeled ternary complexes were halted at a unique template position located upstream of a T-run (TTTTTTTTT) in the nontemplate strand; this element encodes an RNA signal for factor-dependent transcription termination at distal sites on the template. The halted ternary complexes were purified and allowed to resume elongation under a variety of conditions. We found that the T-run constituted a strong elongation block, even at high @entity377 concentrations. The principal sites of pausing were at a C position situated two @entity2036 upstream of the first T in the T-run and at the first three to four T positions within the T-run. There was relatively little pausing at the five downstream Ts. Intrinsic pausing was exacerbated at suboptimal @entity377 concentrations. Ternary complexes arrested by the T-run at 10 microM NTPs rapidly traversed the T-run when the NTP pool was increased to 1 mM. Limiting GTP (1 microM) resulted in polymerase stuttering at the 3' margin of the T-run, immediately prior to a templated G position; this generated a ladder of slippage synthesis products. We found that vaccinia ternary complexes remained intact after elongating to the very end of a linear DNA template and that such complexes catalyzed the addition of extra nucleotides to the 3' end of the RNA chain. The 3' end addition required much higher concentrations of NTPs than did templated chain elongation. Finally, we report that factor-dependent transcription termination by vaccinia RNA polymerase downstream of the T-run was affected by @entity377 concentration. Limiting @entity856 caused the polymerase to terminate at sites closer to the UUUUUNU termination signal. This is consistent with the kinetic coupling model for factor-dependent termination.

Elongation properties of XXXX RNA polymerase: pausing, slippage, 3' end addition, and termination site choice.

@entity856 :: ('MESH:D014544', 'Chemical') :: ['UTP'] @entity2036 :: ('MESH:D009711', 'Chemical') :: ['nucleotides'] @entity377 :: ('CHEBI:36976', 'Chemical') :: ['nucleotide'] @entity6621 :: ('10245', 'Species') :: ['vaccinia virus']
@entity6621 :: (10245,Species) :: ['vaccinia virus']
