Pol II transcription elongation represents a distinct stage within the Pol II transcription cycle (274). During transcription elongation a nascent RNA molecule is synthesized by a stable elongation complex in a processive manner (198). In the course of the last years data has accumulated showing that transcription elongation is extremely complex and highly regulated (196, 274).
1.1.1 Transcript elongation occurs discontinuously and is highly regulated
Studies in higher eukaryotes have shown for selected genes (26, 103) and genome-wide (217, 221, 339) that Pol II pauses 25-50 nt downstream of the TSS at a subset of genes. In
Drosophila cells, promoter proximal pausing was observed at one third of all genes, mainly at
developmentally regulated and stimulus-responsive genes (221). ChIP-chip analyses in yeast grown to stationary phase detected Pol II occupancy at the promoter region of many inactive genes but not downstream of that region (255). Although this observation was interpreted as Pol II pausing, it is not known whether promoter proximal pausing exists in proliferating yeast. Promoter proximal pausing is currently considered as an important regulatory step after the recruitment of Pol II to the gene promoter (196). Particular elongation factors, certain DNA sequences (may be also RNA sequences) and nucleosome occupancy play a role in specifying promoter proximal pausing (220). It is currently under debate whether promoter proximal pausing is a true pausing event of Pol II during transcription elongation or whether the observed occupancy peak of Pol II downstream of the TSS rather represents an intermediate of early termination (David Bentley, University of Colorado, and Stephen Buratowski, Harvard Medical School, personal communication; (288)).
Apart from promoter proximal pausing that usually occurs shortly after the TSS, pausing events can occur throughout the body of genes. ChIP, ChIP-chip and NET-seq studies in yeast could show that Pol II pauses at arrest sites (171), upstream of nucleosomes (49) and at the 3’ end of introns (5, 39). Single cell experiments revealed that the transcript elongation rate
along a gene can vary considerably (62). This is in accord with the observation that transcription occurs in bursts with pulses of high polymerase density (48). All these observations led to the view that transcription elongation is a discontinuous process, interrupted by periods of regulated pauses and arrests. Given the reasonable assumption that several Pol II elongation complexes can transcribe the same gene at a time, pausing would lead to rear-end collisions between the leading elongation complex and the subsequent complexes (267).
1.1.2 Transcription elongation through chromatin
Gene transcription in living cells occurs in a chromatin context (170). Chromatin consists of repeating subunits, the nucleosomes (see also section I 2.2; (191)). Linear nucleosomal arrays represent an extremely strong barrier to Pol II passage (230). One possibility of how Pol II elongation can proceed is nucleosome disassembly in front of Pol II. This mechanism was observed at highly transcribed genes with a high density of Pol II molecules (165, 273). Additionally, a genome-wide analysis of nucleosome occupancy in yeast revealed that the transcription rate is inversely proportional to the histone density in the coding region of genes (178). Although, transcriptionally very active yeast genes were depleted of histones in the coding region, histones immediately re-associated with the DNA at a very high rate when genes were turned off (273). Taken together, during gene transcription the chromatin structure is reversibly altered.
To achieve transcription in a chromatin environment, cells have evolved different classes of factors that together with Pol II are able to modify chromatin structure. Among those factors are chromatin remodelers, histone chaperones, histone modifying enzymes and other transcription factors (13). Whereas chromatin remodelers use the energy of ATP hydrolysis to move, destabilize, eject or restructure nucleosomes (50), histone chaperones assemble or disassemble nucleosomes without using the energy of ATP (13). For example Spt16/Pob3 (also called FACT complex in higher eukaryotes) and Spt6 (see following section) are histone chaperones in yeast that facilitate Pol II transcription elongation in vivo by destabilizing nucleosome structure and by reassembling nucleosomes after Pol II passage (24).
Histone modifying enzymes predominantly modify the flexible histone tails, but also the nucleosomal core (88). Although the best characterized modifications at the moment are histone methylation and acetylation, many other post-translational modifications were known including phosphorylation, ubiquitylation and crotonylation (300). According to the histone code model (292) the post-translational histone modifications are rather used for binding of chromatin-related proteins than directly affecting chromatin compaction by its physicochemical properties. The biological function of the various histone modifications as well as the crosstalk between the different marks is currently far from being understood. Apart from these distinct subsets of factors that alter the chromatin structure other proteins associate with Pol II during transcription elongation to achieve its passage through the nucleosomal DNA template. Among these transcription factors are the Paf1 complex, Spt4/5 (see also section V 1.1), TFIIS, Elf1 and Spn1 (167). Whereas several studies have shed some light on the biological role of the Paf1 complex (142), Spt4/5 (184), and TFIIS (45, 151),
almost nothing is known about Elf1 and Spn1. Elf1 is a non-essential nuclear protein that genetically interacts with Paf1, Spt4/5, Spt6 and Spt16, and localizes to actively transcribed regions (246). Spn1 represents an essential nuclear protein that directly interacts with Spt6 (167). Spn1 may regulates the binding of Spt6 to nucleosomes (200).
Spt6 is a highly conserved nuclear protein and is essential for viability in yeast. Spt6 is required for Pol II transcription elongation and normal chromatin structure. Consistent with its role in transcription elongation, Spt6 co-localizes to chromosomal regions in S. cerevisiae and
Drosophila melanogaster actively transcribed by Pol II. Spt6 stimulates the elongation rate of
Pol II in vivo and interacts with several other transcription elongation factors, including Spt4/5, Elf1 and Spn1. In addition, Spt6 contains a C-terminal tandem SH2 domain that binds phosphorylated Pol II CTD (73, 185, 294) (Figure 5). Deletion of that domain in yeast cells leads to a slow-growth phenotype and is lethal in the presence of 6-azauracil (6-AU), indicating a role in transcription elongation (294). Spt6 may also bind nascent RNA via its S1 domain (Figure 5).
Figure 5: Domain architecture of yeast Spt6. HtH, helix-turn-helix domain, binds double-stranded DNA; YqgFc, predicted to be resolvase or ribonuclease, but in Spt6 catalytic residues are exchanged, thus probably not active; HhH, triple-helix- domain, binding of double stranded DNA; S1, RNA binding domain; SH2-N, SH2-C, tandem SH2 domain; numbers for amino acid residues are indicated; Spt6 is a modular protein with different interaction faces: nucleosome/DNA, RNA and Pol II interaction faces (70). (modified from (294))
Consistent with its role in influencing the chromatin structure, Spt6 directly interacts with histone H3 and can assemble nucleosomes in vitro (32). In agreement with this study, spt6 mutations lead to transcription from cryptic promoters within the coding region of genes, suggesting that Spt6 is required to re-establish correct chromatin structure after Pol II passage (145), especially at highly transcribed genes (133). Spt6 seems also to be involved in the positioning of the +1 nucleosome (133). Recently, it could be shown that Spt6 is required in heterochromatic silencing in Schizosaccaromyces pombe (152).