The initiation-elongation transition: lateral mobility of RNA in RNA polymerase II complexes is greatly reduced at +8/+9 and absent by +23

Abstract

RNA polymerase II transcription complexes stalled shortly after initiation over a repetitive segment of the template can undergo efficient transcript slippage, during which the 3' end of the RNA slides upstream and then re-pairs with the template, allowing transcription to continue. In the present study, we have used transcript slippage as an assay to identify possible structural transitions that occur as the polymerase passes from the initiation to the elongation phase of transcription. We reasoned that transcript slippage would not occur in fully processive complexes. We constructed a series of templates that allowed us to stall RNA polymerase II after the synthesis of a repetitive sequence (5'-CUCUCU-3') at varying distances downstream of +1. We found that polymerase must synthesize at least a 23-nt RNA to attain resistance to transcript slippage. The ability to undergo slippage was lost in two discrete steps, suggestive of two distinct transitions. The first transition is the formation of the 8- to 9-bp mature RNA-DNA hybrid, when slippage abruptly dropped by 10-fold. However, easily detectable slippage continued until 14 more bonds were made. Thus, although the transcript becomes tightly constrained within the transcription complex once the hybrid reaches its final length, much more RNA synthesis is required before the RNA is no longer able to slip upstream along the template. This last point may reflect an important stabilizing role for the interaction of the polymerase with the transcript well upstream of the RNA-DNA hybrid.

Figure 1

Sequences (nontemplate strand) of the initially transcribed regions of the templates used in these studies; note that these templates differ only between the initiating nucleotide (+1) and the first guanine residue (lowercase). The repeated element involved in transcript slippage is shown in italics.

Figure 2

Transcript slippage by pol II is drastically reduced when the polymerase is halted 10 or more bases downstream of +1. Transcription was carried out on the indicated templates (see Fig. 1) with dinucleotide primers in the absence of GTP as described in Experimental Procedures. RNAs in the indicated lanes were digested with RNase T1. Arrowheads designate the expected G-stop (nonslipped) transcripts. Slippage and leakthrough transcripts are marked by asterisks and pound signs, respectively. The percentage slippage is the fraction of complexes that underwent at least one round of transcript slippage. Lengths of selected RNAs are given in the margins of the gel.

Figure 3

(A) TCs initiating with a dinucleotide acquire most of their resistance to transcript slippage on synthesis of a 9-nt transcript. Transcription was carried out on the indicated templates (see Fig. 1) with dinucleotide primers in the absence of GTP as described in Experimental Procedures. RNAs in the indicated lanes were digested with RNase T1. It should be noted that, among the templates we tested, determining the extent of slippage was most problematic with 10G and 11G. All of the T1-resistant bands were scored as slippage products for these templates, although some of these bands do not seem to be the expected length for slippage products. We believe that these anomalous-length RNAs resulted from slippage followed by transcript cleavage from low levels of SII in the reactions, although we have not explored this point further. Pol II transcription complexes may vary considerably in their sensitivity to SII-mediated cleavage (see ref. 18). (B) Incorporation of 5Br-UTP instead of UTP does not significantly alter the transcript slippage activity of halted elongation complexes. Transcription of the indicated templates was performed as in Figs. 2 and 3A except that 5Br-UTP was substituted for UTP in the indicated reactions. An equal aliquot of each reaction mixture was chased with 200 μM of all four NTPs at 30°C for 5 min. Run-off transcripts are not shown. For both panels, arrowheads designate the expected G-stop (nonslipped) transcripts. Slippage and leakthrough transcripts are marked by asterisks and pound signs, respectively. The percentage slippage is the fraction of complexes that underwent at least one round of transcript slippage. Lengths of selected RNAs are given in the margin of the gels.

Figure 4

Slippage activity does not disappear until a 23-nt transcript is synthesized. Transcription was carried out on the indicated templates (see Fig. 1) with ApC primers in the absence of GTP as described in Experimental Procedures. RNAs in the indicated lanes were digested with RNase T1 or chased with 200 μM of all four NTPs. Arrowheads designate the expected G-stop (nonslipped) transcripts. Slippage and leakthrough transcripts are marked by asterisks and pound signs, respectively. The percentage slippage is the fraction of complexes that underwent at least one round of transcript slippage. Lengths of selected RNAs are given in the margin of the gel.

Figure 5

ATP-initiated TCs acquire most of their resistance to transcript slippage on synthesis of an 8-nt RNA. Transcription of the indicated templates was carried as in Fig. 2 except that 50 μM ATP was used instead of a dinucleotide to support initiation. (A) Transcript slippage on templates 6G and 8G. (B) Transcript slippage on templates 8G through 10G. Slippage and leakthrough transcripts are marked by asterisks and pound signs, respectively. The percentage slippage is the fraction of complexes that underwent at least one round of transcript slippage. Lengths of selected RNAs are given in the margins of the gel.

Figure 6

A diagrammatic comparison of transcript slippage and backtracking. The rounded rectangle represents pol II, the paired lines are the two strands of DNA, and the gray boxes are the RNA exit channel and the funnel, respectively. The asterisk is the active site, and the transcript is shown by either the designated dashed (RNA in RNA–DNA hybrid) or solid (RNA not in hybrid) lines. The transcript in this case has a repetitive sequence at the 3′ end, which would allow transcript slippage (steps in left column); an alternative pathway involving backtracking is shown in the right column. Transcript cleavage occurs at step 3 in the backtracking pathway. Resumption of RNA synthesis is shown in step 4 in both cases.