The 8-Nucleotide-long RNA:DNA Hybrid Is a Primary Stability Determinant of the RNA Polymerase II Elongation Complex

Abstract

The sliding clamp model of transcription processivity, based on extensive studies of Escherichia coli RNA polymerase, suggests that formation of a stable elongation complex requires two distinct nucleic acid components: an 8 -9-nt transcript-template hybrid, and a DNA duplex immediately downstream from the hybrid. Here, we address the minimal composition of the processive elongation complex in the eukaryotes by developing a method for promoter-independent assembly of functional elongation complex of S. cerevisiae RNA polymerase II from synthetic DNA and RNA oligonucleotides. We show that only one of the nucleic acid components, the 8-nt RNA: DNA hybrid, is necessary for the formation of a stable elongation complex with RNA polymerase II. The double-strand DNA upstream and downstream of the hybrid does not affect stability of the elongation complex. This finding reveals a significant difference in processivity determinants of RNA polymerase II and E. coli RNA polymerase. In addition, using the imperfect RNA:DNA hybrid disturbed by the mismatches in the RNA, we show that nontemplate DNA strand may reduce the elongation complex stability via the reduction of the RNA:DNA hybrid length. The structure of a "minimal stable" elongation complex suggests a key role of the RNA:DNA hybrid in RNA polymerase II processivity.Characterization of the processivity determinants in yeast RNA polymerase II (Pol II) 1 is crucial for understanding the mechanisms controlling eukaryotic gene expression at the levels of promoter escape, pausing, arrest, and release of the RNA from transcription terminators (1, 2). The polymerase proceeds through the nucleosomal structure of the template and survives a prolonged pausing or arrest in the genes without dissociating from the template. Therefore, the formation of a highly stable elongation complex (EC), in which RNA polymerase is tightly bound to the nascent transcript and template, is absolutely required for the enzyme processivity (3). The mechanism that reconciles the strong stable binding of Pol II to the DNA with the high speed of forward translocation is unknown.Elongation of a promoter-initiated transcript occurs in the absence of general initiation factors, which dissociate from the enzyme during promoter escape (4). Processive elongation by eukaryotic Pol II can also be achieved using purified polymerase in a promoter-and factor-independent transcription system (5). Therefore, it is likely that the basic processivity function belongs to the core Pol II enzyme. Although it was shown that substantial changes in the nucleic acid array accompany the switch to a processive RNA synthesis (6), the role of DNA and RNA in the Pol II EC stability remains speculative (7). The elucidation of this role has been hampered by the extreme complexity of the native eukaryotic EC, which contains multiple transcription elongation factors (8). Differentiation between the effect of elongation factors on EC stability and activity and the role of the Pol II core enzyme interaction with RNA and DNA requires a...

Transcription termination by nuclear RNA polymerases

“…Depletion of Rat1 inhibits but does not abolish termination (Kim et al 2004b), and digestion of RNAPII-associated RNA is not sufficient to disassemble the EC in vitro Kireeva et al 2000). Consistent with this, the abundant and mostly cytoplasmic exonuclease Xrn1, which is similar to Rat1, is able to complement the exonuclease activity of a Rat1-deficient strain when targeted to the nucleus, but unable to complement the termination defect (Luo et al 2006).…”

Characterization of T7 RNA Polymerase Transcription Complexes Assembled on Nucleic Acid Scaffolds

“…2 that the presence of the NT strand is not required to provide salt resistance to T7 RNAP complexes is in contrast to previous findings with E. coli RNA polymerase RNAP (24) but not with yeast polymerase II (37). To examine the contribution of the downstream DNA in more detail, we varied the length of the downstream DNA both in the presence and in the absence of a complementary NT strand (Fig.…”

Histone H3.3 promotes IgV gene diversification by enhancing formation of AID ‐accessible single‐stranded DNA

“…AID is able to access the ssDNA generated during transcription by bacteriophage T7 polymerase in vitro but does so in a very strand‐biased manner, predominantly targeting the looped‐out non‐template strand (Chaudhuri et al , 2003). However, during transcription in vivo this loop within the elongating transcription bubble is short, corresponding to an R‐loop of 8 base pairs (Kireeva et al , 2000), and is likely to be quite well protected within the RNAPII complex. It thus may not be readily accessible to AID under normal circumstances.…”

“…The length of the single‐stranded DNA patches was determined by counting the distance, in nucleotides, between the two most distant consecutive dCs that have been converted to dTs (for ssDNA in the coding strand, read as dGs to dAs for the template strand), including these converted nucleotides and all the non‐dCs (dGs for the template strand) enclosed within the segment. Since the normal transcription bubble is thought to be 8 nt in length (Kireeva et al , 2000), only segments of a length of ≥ 8 nucleotides were included in the analysis. The plot of dG and dC content along IGVL R was created using the GC Content Calculator tool (http://www.biologicscorp.com/tools/GCContent/), with a window size of 30 nucleotides.…”