Insights into how Spt5 functions in transcription elongation and repressing transcription coupled DNA repair.
ABSTRACT: Spt5, a transcription elongation factor, and Rpb4, a subunit of RNA polymerase II (RNAP II) that forms a subcomplex with Rpb7, play important roles in transcription elongation and repression of transcription coupled DNA repair (TCR) in eukaryotic cells. How Spt5 physically interacts with RNAP II, and if and/or how Spt5 and Rpb4/7 coordinate to achieve the distinctive functions have been enigmatic. By site-specific incorporation of the unnatural amino acid p-benzoyl-L-phenylalanine, a photoreactive cross-linker, we mapped interactions between Spt5 and RNAP II in Saccharomyces cerevisiae. Through its KOW4-5 domains, Spt5 extensively interacts with Rpb4/7. Spt5 also interacts with Rpb1 and Rpb2, two largest subunits of RNAP II, at the clamp, protrusion and wall domains. These interactions may lock the clamp to the closed conformation and enclose the DNA being transcribed in the central cleft of RNAP II. Deletion of Spt5 KOW4-5 domains decreases transcription elongation and derepresses TCR. Our findings suggest that Spt5 is a key coordinator for holding the RNAP II complex in a closed conformation that is highly competent for transcription elongation but repressive to TCR.
Project description:The human transcription elongation factor DSIF is highly conserved throughout all kingdoms of life and plays multiple roles during transcription. DSIF is a heterodimer, consisting of Spt4 and Spt5 that interacts with RNA polymerase II (RNAP II). DSIF binds to the elongation complex and induces promoter-proximal pausing of RNAP II. Human Spt5 consists of a NusG N-terminal (NGN) domain motif, which is followed by several KOW domains. We determined the solution structures of the human Spt5 KOW4 and the C-terminal domain by nuclear magnetic resonance spectroscopy. In addition to the typical KOW fold, the solution structure of KOW4 revealed an N-terminal four-stranded ?-sheet, previously designated as the KOW3-KOW4 linker. In solution, the C-terminus of Spt5 consists of two ?-barrel folds typical for KOW domains, designated KOW6 and KOW7. We also analysed the nucleic acid and RNAP II binding properties of the KOW domains. KOW4 variants interacted with nucleic acids, preferentially single stranded RNA, whereas no nucleic acid binding could be detected for KOW6-7. Weak binding of KOW4 to the RNAP II stalk, which is comprised of Rpb4/7, was also detected, consistent with transient interactions between Spt5 and these RNAP II subunits.
Project description:Related RNA polymerases (RNAPs) carry out cellular gene transcription in all three kingdoms of life. The universal conservation of the transcription machinery extends to a single RNAP-associated factor, Spt5 (or NusG in bacteria), which renders RNAP processive and may have arisen early to permit evolution of long genes. Spt5 associates with Spt4 to form the Spt4/5 heterodimer. Here, we present the crystal structure of archaeal Spt4/5 bound to the RNAP clamp domain, which forms one side of the RNAP active centre cleft. The structure revealed a conserved Spt5-RNAP interface and enabled modelling of complexes of Spt4/5 counterparts with RNAPs from all kingdoms of life, and of the complete yeast RNAP II elongation complex with bound Spt4/5. The N-terminal NGN domain of Spt5/NusG closes the RNAP active centre cleft to lock nucleic acids and render the elongation complex stable and processive. The C-terminal KOW1 domain is mobile, but its location is restricted to a region between the RNAP clamp and wall above the RNA exit tunnel, where it may interact with RNA and/or other factors.
Project description:Spt5 is the only known RNA polymerase-associated factor that is conserved in all three domains of life. We have solved the structure of the Methanococcus jannaschii Spt4/5 complex by X-ray crystallography, and characterized its function and interaction with the archaeal RNAP in a wholly recombinant in vitro transcription system. Archaeal Spt4 and Spt5 form a stable complex that associates with RNAP independently of the DNA-RNA scaffold of the elongation complex. The association of Spt4/5 with RNAP results in a stimulation of transcription processivity, both in the absence and the presence of the non-template strand. A domain deletion analysis reveals the molecular anatomy of Spt4/5--the Spt5 Nus-G N-terminal (NGN) domain is the effector domain of the complex that both mediates the interaction with RNAP and is essential for its elongation activity. Using a mutagenesis approach, we have identified a hydrophobic pocket on the Spt5 NGN domain as binding site for RNAP, and reciprocally the RNAP clamp coiled-coil motif as binding site for Spt4/5.
Project description:Evolutionary related multisubunit RNA polymerases from all three domains of life, Eukarya, Archaea and Bacteria, have common structural and functional properties. We have recently shown that two RNAP subunits, F/E (RPB4/7)-which are conserved between eukaryotes and Archaea but have no bacterial homologues-interact with the nascent RNA chain and thereby profoundly modulate RNAP activity. Overall F/E increases transcription processivity, but it also stimulates transcription termination in a sequence-dependent manner. In addition to RNA-binding, these two apparently opposed processes are likely to involve an allosteric mechanism of the RNAP clamp. Spt4/5 is the only known RNAP-associated transcription factor that is conserved in all three domains of life, and it stimulates elongation similar to RNAP subunits F/E. Spt4/5 enhances processivity in a fashion that is independent of the nontemplate DNA strand, by interacting with the RNAP clamp. Whereas the molecular mechanism of Spt4/5 is universally conserved in evolution, the added functionality of F/E-like complexes has emerged after the split of the bacterial and archaeoeukaryotic lineages. Interestingly, bacteriophage-encoded antiterminator proteins could, in theory, fulfil an analogous function in the bacterial RNAP.
Project description:Termination of RNA polymerase II (Pol II) transcription is a key step that is important for 3' end formation of functional mRNA, mRNA release, and Pol II recycling. Even so, the underlying termination mechanism is not yet understood. Here, we demonstrate that the conserved and essential termination factor Seb1 is found on Pol II near the end of the RNA exit channel and the Rpb4/7 stalk. Furthermore, the Seb1 interaction surface with Pol II largely overlaps with that of the elongation factor Spt5. Notably, Seb1 co-transcriptional recruitment is dependent on Spt5 dephosphorylation by the conserved PP1 phosphatase Dis2, which also dephosphorylates threonine 4 within the Pol II heptad repeated C-terminal domain. We propose that Dis2 orchestrates the transition from elongation to termination phase during the transcription cycle by mediating elongation to termination factor exchange and dephosphorylation of Pol II C-terminal domain.
Project description:Biogenesis of messenger RNA is critically influenced by the phosphorylation state of the carboxy-terminal domain (CTD) in the largest RNA polymerase II (RNAPII) subunit. Several kinases and phosphatases are required to maintain proper CTD phosphorylation levels and, additionally, several other proteins modulate them, including Rpb4/7 and Sub1. The Rpb4/7 heterodimer, constituting the RNAPII stalk, promote phosphatase functions and Sub1 globally influences CTD phosphorylation, though its mechanism remains mostly unknown. Here, we show that Sub1 physically interacts with the RNAPII stalk domain, Rpb4/7, likely through its C-terminal region, and associates with Fcp1. While Rpb4 is not required for Sub1 interaction with RNAPII complex, a fully functional heterodimer is required for Sub1 association to promoters. We also demonstrate that a complete CTD is necessary for proper association of Sub1 to chromatin and to the RNAPII. Finally, genetic data show a functional relationship between Sub1 and the RNAPII clamp domain. Altogether, our results indicate that Sub1, Rpb4/7 and Fcp1 interaction modulates CTD phosphorylation. In addition, Sub1 interaction with Rpb4/7 can also modulate transcription start site selection and transcription elongation rate likely by influencing the clamp function.
Project description:The transcriptional coactivator Sub1 has been implicated in several steps of mRNA metabolism in yeast, such as the activation of transcription, termination, and 3'-end formation. In addition, Sub1 globally regulates RNA polymerase II phosphorylation, and most recently it has been shown that it is a functional component of the preinitiation complex. Here we present evidence that Sub1 plays a significant role in transcription elongation by RNA polymerase II (RNAPII). We show that SUB1 genetically interacts with the gene encoding the elongation factor Spt5, that Sub1 influences Spt5 phosphorylation of the carboxy-terminal domain of RNAPII largest subunit by the kinase Bur1, and that both Sub1 and Spt5 copurify in the same complex, likely during early transcription elongation. Indeed, our data indicate that Sub1 influences Spt5-Rpb1 interaction. In addition, biochemical and molecular data show that Sub1 influences transcription elongation of constitutive and inducible genes and associates with coding regions in a transcription-dependent manner. Taken together, our results indicate that Sub1 associates with Spt5 and influences Spt5-Rpb1 complex levels and consequently transcription elongation rate.
Project description:In all domains of life, elongating RNA polymerases require the assistance of accessory factors to maintain their processivity and regulate their rate. Among these elongation factors, the Spt5/NusG factors stand out. Members of this protein family appear to be the only transcription accessory proteins that are universally conserved across all domains of life. In archaea and eukaryotes, Spt5 associates with a second protein, Spt4. In addition to regulating elongation, the eukaryotic Spt4-Spt5 complex appears to couple chromatin modification states and RNA processing to transcription elongation. This review discusses the experimental bases for our current understanding of Spt4-Spt5 function and recent studies that are beginning to elucidate the structure of Spt4-Spt5/RNA polymerase complexes and mechanism of Spt4-Spt5 action. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.
Project description:Transcription elongation in vitro is affected by the interactions between RNA polymerase (RNAP) subunits and the nucleic acid scaffold of the ternary elongation complex (TEC, RNAP-DNA-RNA). We have investigated the role of the RNAP subunits F/E (homologous to eukaryotic RPB4/7) during transcription elongation and termination using a wholly recombinant archaeal RNAP and synthetic nucleic acid scaffolds. The F/E complex greatly stimulates the processivity of RNAP, it enhances the formation of full length products, reduces pausing, and increases transcription termination facilitated by weak termination signals. Mutant variants of F/E that are defective in RNA binding show that these activities correlate with the nucleic acid binding properties of F/E. However, a second RNA-binding independent component also contributes to the stimulatory activities of F/E. In summary, our results suggest that interactions between RNAP subunits F/E and the RNA transcript are pivotal to the molecular mechanisms of RNAP during transcription elongation and termination.
Project description:NusG/RfaH/Spt5 transcription elongation factors are the only transcription regulators conserved across all life. Bacterial NusG regulates RNA polymerase (RNAP) elongation complexes (ECs) across most genes, enhancing elongation by suppressing RNAP backtracking and coordinating ?-dependent termination and translation. The NusG paralog RfaH engages the EC only at operon polarity suppressor (ops) sites and suppresses both backtrack and hairpin-stabilized pausing. We used single-particle cryoelectron microscopy (cryo-EM) to determine structures of ECs at ops with NusG or RfaH. Both factors chaperone base-pairing of the upstream duplex DNA to suppress backtracking, explaining stimulation of elongation genome-wide. The RfaH-opsEC structure reveals how RfaH confers operon specificity through specific recognition of an ops hairpin in the single-stranded nontemplate DNA and tighter binding to the EC to exclude NusG. Tight EC binding by RfaH sterically blocks the swiveled RNAP conformation necessary for hairpin-stabilized pausing. The universal conservation of NusG/RfaH/Spt5 suggests that the molecular mechanisms uncovered here are widespread.