A model in vitro system for co-transcriptional splicing

A hallmark of metazoan RNA polymerase II transcripts is the presence of numerous small exons surrounded by large introns. Abundant evidence indicates that splicing to excise introns occurs co-transcriptionally, prior to release of the nascent transcript from RNAP II. Here, we established an efficient model system for co-transcriptional splicing in vitro. In this system, CMV-DNA constructs immobilized on beads generate RNAP II transcripts containing two exons and an intron. Consistent with previous work, our data indicate that elongating nascent transcripts are tethered to RNAP II on the immobilized DNA template. We show that nascent transcripts that reach full length, but are still attached to RNAP II, are efficiently spliced. When the nascent transcript is cleaved within the intron using RNase H, both the 5′ and 3′ cleavage fragments are detected in the bound fraction, where they undergo splicing. Together, our work establishes a system for co-transcriptional splicing in vitro, in which the spliceosome containing the 5′ and 3′ exons are tethered to RNAP II for splicing

Rearrangement of the RNA polymerase subunit H and the lower jaw in archaeal elongation complexes

The lower jaws of archaeal RNA polymerase and eukaryotic RNA polymerase II include orthologous subunits H and Rpb5, respectively. The tertiary structure of H is very similar to the structure of the C-terminal domain of Rpb5, and both subunits are proximal to downstream DNA in pre-initiation complexes. Analyses of reconstituted euryarchaeal polymerase lacking subunit H revealed that H is important for open complex formation and initial transcription. Eukaryotic Rpb5 rescues activity of the ΔH enzyme indicating a strong conservation of function for this subunit from archaea to eukaryotes. Photochemical cross-linking in elongation complexes revealed a striking structural rearrangement of RNA polymerase, bringing subunit H near the transcribed DNA strand one helical turn downstream of the active center, in contrast to the positioning observed in preinitiation complexes. The rearrangement of subunits H and A′′ suggest a major conformational change in the archaeal RNAP lower jaw upon formation of the elongation complex

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

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