RNA chain elongation by RNA polymerase II (pol II) is a complex and regulated process which is coordinated with capping, splicing, and polyadenylation of the primary transcript. Numerous elongation factors that enable pol II to transcribe faster and/or more efficiently have been purified. SII is one such factor. It helps pol II bypass specific blocks to elongation that are encountered during transcript elongation. SII was first identified biochemically on the basis of its ability to enable pol II to synthesize long transcripts.(1) Both the high resolution structure of SII and the details of its novel mechanism of action have been refined through mutagenesis and sophisticated in vitro assays. SII engages transcribing pol II and assists it in bypassing blocks to elongation by stimulating a cryptic, nascent RNA cleavage activity intrinsic to RNA polymerase. The nuclease activity can also result in removal of misincorporated bases from RNA. Molecular genetic experiments in yeast suggest that SII is generally involved in mRNA synthesis in vivo and that it is one type of a growing collection of elongation factors that regulate pol II. In vertebrates, a family of related SII genes has been identified; some of its members are expressed in a tissue‐specific manner. The principal challenge now is to understand the isoform‐specific functional differences and the biology of regulation exerted by the SII family of proteins on target genes, particularly in multicellular organisms. BioEssays 22:327–336, 2000. © 2000 John Wiley & Sons, Inc.

Elongation factor SII interacts with RNA polymerase II and enables it to transcribe through arrest sites in vitro. The set of genes dependent upon SII function in vivo and the effects on RNA levels of mutations in different components of the elongation machinery are poorly understood. Using yeast lacking SII and bearing a conditional allele of RPB2, the gene encoding the second largest subunit of RNA polymerase II, we describe a genetic interaction between SII and RPB2. An SII gene disruption or the rpb2-10 mutation, which yields an arrest-prone enzyme in vitro, confers sensitivity to 6-azauracil (6AU), a drug that depresses cellular nucleoside triphosphates. Cells with both mutations had reduced levels of total poly(A) ؉ RNA and specific mRNAs and displayed a synergistic level of drug hypersensitivity. In cells in which the SII gene was inactivated, rpb2-10 became dominant, as if template-associated mutant RNA polymerase II hindered the ability of wild-type polymerase to transcribe. Interestingly, while 6AU depressed RNA levels in both wild-type and mutant cells, wild-type cells reestablished normal RNA levels, whereas double-mutant cells could not. This work shows the importance of an optimally functioning elongation machinery for in vivo RNA synthesis and identifies an initial set of candidate genes with which SII-dependent transcription can be studied.The elongation phase of transcription is an important control point for the regulation of gene expression (reviewed in references 4, 29, and 41). Several general elongation factors, including TFIIF, ELL, and elongin (SIII), are able to increase the overall rate of transcription elongation of RNA polymerase II (PolII) in vitro (7,13,26,37). SII enables PolII to transcribe through a variety of transcriptional blockages, including intrinsic arrest sites and nucleoprotein complexes (reviewed in references 29 and 41). SII reactivates arrested PolII complexes by binding to the enzyme and activating a nascent RNA cleavage activity, which eventually results in polymerase escape (reviewed in reference 28). It has been hypothesized that elongation factors such as TFIIF reduce the frequency of arrest by reducing the dwell time of PolII at arrest sites (6,15).Little is known about gene sequences that block transcription and the interaction of general elongation factors with PolII complexes in vivo. In yeast, mutation or disruption of the gene encoding SII, DST1 (gene names in this study are those designated in the Saccharomyces Genome Database; DST1 is also known as PPR2), renders cells sensitive to the base analog 6-azauracil (6AU) (19,23,24). 6AU depletes cellular levels of the RNA precursors UTP and GTP (12). Supplementing the drug-containing medium with uracil or guanine reverses this phenotype, suggesting that the drug inhibits growth because of the reduction in nucleoside triphosphate levels and thereby impairs transcription elongation (3, 12). The sensitivity of yeast to 6AU after disruption of DST1 may indicate an increased requirement by RNA polymerase for elongat...

RNA polymerases encounter specific DNA sites at which RNA chain elongation takes place in the absence of enzyme translocation in a process called discontinuous elongation. For RNA polymerase II, at least some of these sequences also provoke transcriptional arrest where renewed RNA polymerization requires elongation factor SII. Recent elongation models suggest the occupancy of a site within RNA polymerase that accommodates nascent RNA during discontinuous elongation. Here we have probed the extent of nascent RNA extruded from RNA polymerase II as it approaches, encounters, and departs an arrest site. Just upstream of an arrest site, 17-19 nucleotides of the RNA 3'-end are protected from exhaustive digestion by exogenous ribonuclease probes. As RNA is elongated to the arrest site, the enzyme does not translocate and the protected RNA becomes correspondingly larger, up to 27 nucleotides in length. After the enzyme passes the arrest site, the protected RNA is again the 18-nucleotide species typical of an elongation-competent complex. These findings identify an extended RNA product groove in arrested RNA polymerase II that is probably identical to that emptied during SII-activated RNA cleavage, a process required for the resumption of elongation. Unlike Escherichia coli RNA polymerase at a terminator, arrested RNA polymerase II does not release its RNA but can reestablish the normal elongation mode downstream of an arrest site. Discontinuous elongation probably represents a structural change that precedes, but may not be sufficient for, arrest by RNA polymerase II.Recent studies of the transcription elongation process have revealed that RNA chain synthesis is more complex than previously appreciated (1-8). At specific template sequences, RNA chain elongation by bacterial or eukaryotic RNA polymerases is uncoupled from translocation on DNA (1-8). The fraction of total transcription that takes place in this discontinuous or inchworm-like mode is unclear. The changing relationship between the RNA 3'-end and the elongation complex's DNA contacts has been mapped for a large collection of complexes at different positions on a single template (4). This survey suggested that inchworming alternates with periods of smooth chain assembly; during the latter process, base incorporation is coupled to translocation. It would appear that discontinuous elongation is provoked by specific DNA sequences and is accompanied by an unusual positioning of the RNA 3' terminus close to the complex's downstream boundary (3,4). This has been described as a "strained" (energetically unfavorable) configuration (4, 9, 10). Indeed, acquisition of the strained conformation appears to prepare the Escherichia coli elongation complex for termination (6, 7).Models of discontinuous elongation include an RNA product site in RNA polymerase that is alternately filled and emptied (5, 10, 11). An RNA-binding site has been observed on bacterial and eukaryotic RNA polymerases in which the RNA 3'-end is positioned near the enzyme's active site (12,13). In...