ELONGATION PROPERTIES OF VACCINIA VIRUS-RNA POLYMERASE - PAUSING, SLIPPAGE, 3'-END ADDITION, AND TERMINATION SITE CHOICE

We have analyzed the elongation properties of vaccinia virus RNA polym erase during a single round of transcription in vitro. RNA-labeled ter nary complexes were halted at a unique template position located upstr eam of a T-run (TTTTTTTTT) in the nontemplate strand; this element enc odes an RNA signal for factor-dependent transcription termination at d istal sites on the template. The halted ternary complexes were purifie d and allowed to resume elongation under a variety of conditions. We f ound that the T-run constituted a strong elongation block, even at hig h nucleotide concentrations. The principal sites of pausing were at a C position situated two nucleotides upstream of the first T in the T-r un and at the first three to four T positions within the T-run. There was relatively little pausing at the five downstream Ts. intrinsic pau sing was exacerbated at suboptimal nucleotide concentrations. Ternary complexes arrested by the T-run at 10 mu M NTPs rapidly traversed the T-run when the NTP pool was increased to 1 mM. Limiting GTP (1 mu M) r esulted in polymerase stuttering at the 3' margin of the T-run, immedi ately prior to a templated G position; this generated a ladder of slip page synthesis products. We found that vaccinia ternary complexes rema ined 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 nucleotide conc entration. Limiting UTP caused the polymerase to terminate at sites cl oser to the UUUUUNU termination signal. This is consistent with the ki netic coupling model for factor-dependent termination.