Structures and Functions of the Multiple KOW Domains of Transcription Elongation Factor Spt5.
ABSTRACT: The eukaryotic Spt4-Spt5 heterodimer forms a higher-order complex with RNA polymerase II (and I) to regulate transcription elongation. Extensive genetic and functional data have revealed diverse roles of Spt4-Spt5 in coupling elongation with chromatin modification and RNA-processing pathways. A mechanistic understanding of the diverse functions of Spt4-Spt5 is hampered by challenges in resolving the distribution of functions among its structural domains, including the five KOW domains in Spt5, and a lack of their high-resolution structures. We present high-resolution crystallographic results demonstrating that distinct structures are formed by the first through third KOW domains (KOW1-Linker1 [K1L1] and KOW2-KOW3) of Saccharomyces cerevisiae Spt5. The structure reveals that K1L1 displays a positively charged patch (PCP) on its surface, which binds nucleic acids in vitro, as shown in biochemical assays, and is important for in vivo function, as shown in growth assays. Furthermore, assays in yeast have shown that the PCP has a function that partially overlaps that of Spt4. Synthesis of our results with previous evidence suggests a model in which Spt4 and the K1L1 domain of Spt5 form functionally overlapping interactions with nucleic acids upstream of the transcription bubble, and this mechanism may confer robustness on processes associated with transcription elongation.
Project description:The Spt4-Spt5 complex is an essential RNA polymerase II elongation factor found in all eukaryotes and important for gene regulation. We report here the crystal structure of Saccharomyces cerevisiae Spt4 bound to the NGN domain of Spt5. This structure reveals that Spt4-Spt5 binding is governed by an acid-dipole interaction between Spt5 and Spt4. Mutations that disrupt this interaction disrupt the complex. Residues forming this pivotal interaction are conserved in the archaeal homologs of Spt4 and Spt5, which we show also form a complex. Even though bacteria lack a Spt4 homolog, the NGN domains of Spt5 and its bacterial homologs are structurally similar. Spt4 is located at a position that may help to maintain the functional conformation of the following KOW domains in Spt5. This structural and evolutionary perspective of the Spt4-Spt5 complex and its homologs suggest that it is an ancient, core component of the transcription elongation machinery.
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:The heterodimeric transcription elongation factor Spt4/Spt5 (Spt4/5) tightly associates with RNAPII to regulate both transcriptional elongation and co-transcriptional pre-mRNA processing; however, the mechanisms by which Spt4/5 acts are poorly understood. Recent studies of the human and Drosophila Spt4/5 complexes indicate that they can bind nucleic acids in vitro. We demonstrate here that yeast Spt4/5 can bind in a sequence-specific manner to single stranded RNA containing AAN repeats. Furthermore, we show that the major protein determinants for RNA-binding are Spt4 together with the NGN domain of Spt5 and that the KOW domains are not required for RNA recognition. These findings attribute a new function to a domain of Spt4/5 that associates directly with RNAPII, making significant steps towards elucidating the mechanism behind transcriptional control by Spt4/5.
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:During transcription elongation, eukaryotic RNA polymerase II (Pol II) must contend with the barrier presented by nucleosomes. The conserved Spt4-Spt5 complex has been proposed to regulate elongation through nucleosomes by Pol II. To help define the mechanism of Spt5 function, we have characterized proteins that coimmunopurify with Spt5. Among these are the general elongation factors TFIIF and TFIIS as well as Spt6 and FACT, factors thought to regulate elongation through nucleosomes. Spt5 also coimmunopurified with the mRNA capping enzyme and cap methyltransferase, and spt4 and spt5 mutations displayed genetic interactions with mutations in capping enzyme genes. Additionally, we found that spt4 and spt5 mutations lead to accumulation of unspliced pre-mRNA. Spt5 also copurified with several previously unstudied proteins; we demonstrate that one of these is encoded by a new member of the SPT gene family. Finally, by immunoprecipitating these factors we found evidence that Spt5 participates in at least three Pol II complexes. These observations provide new evidence of roles for Spt4-Spt5 in pre-mRNA processing and transcription elongation.
Project description:Previous characterization of the Saccharomyces cerevisiae Spt4, Spt5, and Spt6 proteins suggested that these proteins act as transcription factors that modify chromatin structure. In this work, we report new genetic and biochemical studies of Spt4, Spt5, and Spt6 that reveal a role for these factors in transcription elongation. We have isolated conditional mutations in SPT5 that can be suppressed in an allele-specific manner by mutations in the two largest subunits of RNA polymerase II (Pol II). Strikingly, one of these RNA Pol II mutants is defective for transcription elongation and the others cause phenotypes consistent with an elongation defect. In addition, we show that spt4, spt5, and spt6 mutants themselves have phenotypes suggesting defects in transcription elongation in vivo. Consistent with these findings, we show that Spt5 is physically associated with RNA Pol II in vivo, and have identified a region of sequence similarity between Spt5 and NusG, an Escherichia coli transcription elongation factor that binds directly to RNA polymerase. Finally, we show that Spt4 and Spt5 are tightly associated in a complex that does not contain Spt6. These results, taken together with the biochemical identification of a human Spt4-Spt5 complex as a transcription elongation factor (Wada et al. 1998), provide strong evidence that these factors are important for transcription elongation in vivo.
Project description:The heterodimeric complex SPT4/SPT5 is a transcript elongation factor (TEF) that directly interacts with RNA polymerase II (RNAPII) to regulate messenger RNA synthesis in the chromatin context. We provide biochemical evidence that in Arabidopsis, SPT4 occurs in a complex with SPT5, demonstrating that the SPT4/SPT5 complex is conserved in plants. Each subunit is encoded by two genes SPT4-1/2 and SPT5-1/2. A mutant affected in the tissue-specifically expressed SPT5-1 is viable, whereas inactivation of the generally expressed SPT5-2 is homozygous lethal. RNAi-mediated downregulation of SPT4 decreases cell proliferation and causes growth reduction and developmental defects. These plants display especially auxin signalling phenotypes. Consistently, auxin-related genes, most strikingly AUX/IAA genes, are downregulated in SPT4-RNAi plants that exhibit an enhanced auxin response. In Arabidopsis nuclei, SPT5 clearly localizes to the transcriptionally active euchromatin, and essentially co-localizes with transcribing RNAPII. Typical for TEFs, SPT5 is found over the entire transcription unit of RNAPII-transcribed genes. In SPT4-RNAi plants, elevated levels of RNAPII and SPT5 are detected within transcribed regions (including those of downregulated genes), indicating transcript elongation defects in these plants. Therefore, SPT4/SPT5 acts as a TEF in Arabidopsis, regulating transcription during the elongation stage with particular impact on the expression of certain auxin-related genes.
Project description:The Spt5-Spt4 complex regulates early transcription elongation by RNA polymerase II and has an imputed role in pre-mRNA processing via its physical association with mRNA capping enzymes. Here we characterize the Schizosaccharomyces pombe core Spt5-Spt4 complex as a heterodimer and map a trypsin-resistant Spt4-binding domain within the Spt5 subunit. A genetic analysis of Spt4 in S. pombe revealed it to be inessential for growth at 25 degrees C-30 degrees C but critical at 37 degrees C. These results echo the conditional spt4Delta growth phenotype in budding yeast, where we find that Saccharomyces cerevisiae and S. pombe Spt4 are functionally interchangeable. Complementation of S. cerevisiae spt4Delta and a two-hybrid assay for Spt4-Spt5 interaction provided a readout of the effects of 33 missense and truncation mutations on S. pombe Spt4 function in vivo, which were interpreted in light of the recent crystal structure of S. cerevisiae Spt4 fused to a fragment of Spt5. Our results highlight the importance of the Spt4 Zn2+-binding residues--Cys12, Cys15, Cys29, and Asp32--and of Ser57, a conserved constituent of the Spt4-Spt5 interface. The 990-amino acid S. pombe Spt5 protein has an exceptionally regular carboxyl-terminal domain (CTD) composed of 18 nonapeptide repeats. We find that as few as three nonamer repeats sufficed for S. pombe growth, but only when Spt4 was present. Synthetic lethality of the spt5(1-835) spt4Delta double mutant at 34 degrees C suggests that interaction of Spt4 with the central domain of Spt5 overlaps functionally with the Spt5 CTD.
Project description:Spt5 is a transcription factor conserved in all three domains of life. Spt5 homologues from bacteria and archaea bind the largest subunit of their respective RNA polymerases. Here we demonstrate that Spt5 directly associates with RNA polymerase (Pol) I and RNA Pol II in yeast through its central region containing conserved NusG N-terminal homology and KOW domains. Deletion analysis of SPT5 supports our biochemical data, demonstrating the importance of the KOW domains in Spt5 function. Far Western blot analysis implicates A190 of Pol I as well as Rpb1 of Pol II in binding Spt5. Three additional subunits of Pol I may also participate in this interaction. One of these subunits, A49, has known roles in transcription elongation by Pol I. Interestingly, spt5 truncation mutations suppress the cold-sensitive phenotype of rpa49? strain, which lacks the A49 subunit in the Pol I complex. Finally, we observed that Spt5 directly binds to an essential Pol I transcription initiation factor, Rrn3, and to the ribosomal RNA. Based on these data, we propose a model in which Spt5 is recruited to the rDNA early in transcription and propose that it plays an important role in ribosomal RNA synthesis through direct binding to the Pol I complex.
Project description:RNA polymerase II (RNAPII) undergoes structural changes during the transitions from initiation, elongation, and termination, which are aided by a collection of proteins called elongation factors. NusG/Spt5 is the only elongation factor conserved in all domains of life. Although much information exists about the interactions between NusG/Spt5 and RNA polymerase in prokaryotes, little is known about how the binding of eukaryotic Spt4/5 affects the biochemical activities of RNAPII. We characterized the activities of Spt4/5 and interrogated the structural features of Spt5 required for it to interact with elongation complexes, bind nucleic acids, and promote transcription elongation. The eukaryotic specific regions of Spt5 containing the Kyrpides, Ouzounis, Woese domains are involved in stabilizing the association with the RNAPII elongation complex, which also requires the presence of the nascent transcript. Interestingly, we identify a region within the conserved NusG N-terminal (NGN) domain of Spt5 that contacts the non-template strand of DNA both upstream of RNAPII and in the transcription bubble. Mutating charged residues in this region of Spt5 did not prevent Spt4/5 binding to elongation complexes, but abrogated the cross-linking of Spt5 to DNA and the anti-arrest properties of Spt4/5, thus suggesting that contact between Spt5 (NGN) and DNA is required for Spt4/5 to promote elongation. We propose that the mechanism of how Spt5/NGN promotes elongation is fundamentally conserved; however, the eukaryotic specific regions of the protein evolved so that it can serve as a platform for other elongation factors and maintain its association with RNAPII as it navigates genomes packaged into chromatin.