Structural insight into recognition of phosphorylated threonine-4 of RNA polymerase II C-terminal domain by Rtt103p

Phosphorylation patterns of the C-terminal domain (CTD) of largest subunit of RNA polymerase II (called the CTD code) orchestrate the recruitment of RNA processing and transcription factors. Recent studies showed that not only serines and tyrosines but also threonines of the CTD can be phosphorylated with a number of functional consequences, including the interaction with yeast transcription termination factor, Rtt103p. Here, we report the solution structure of the Rtt103p CTD-interacting domain (CID) bound to Thr4 phosphorylated CTD, a poorly understood letter of the CTD code. The structure reveals a direct recognition of the phospho-Thr4 mark by Rtt103p CID and extensive interactions involving residues from three repeats of the CTD heptad. Intriguingly, Rtt103p's CID binds equally well Thr4 and Ser2 phosphorylated CTD. A doubly phosphorylated CTD at Ser2 and Thr4 diminishes its binding affinity due to electrostatic repulsion. Our structural data suggest that the recruitment of a CID-containing CTD-binding factor may be coded by more than one letter of the CTD code

Structure and dynamics of the RNAPII CTDsome with Rtt103

RNA polymerase II (RNAPII) not only transcribes protein coding genes and many noncoding RNA, but also coordinates transcription and RNA processing. This coordination is mediated by a long C-terminal domain (CTD) of the largest RNAPII subunit, which serves as a binding platform for many RNA/protein-binding factors involved in transcription regulation. In this work, we used a hybrid approach to visualize the architecture of the full-length CTD in complex with the transcription termination factor Rtt103. Specifically, we first solved the structures of the isolated subcomplexes at high resolution and then arranged them into the overall envelopes determined at low resolution by small-angle X-ray scattering. The reconstructed overall architecture of the Rtt103–CTD complex reveals how Rtt103 decorates the CTD platform

Termination of non-coding transcription in yeast relies on both an RNA Pol II CTD interaction domain and a CTD-mimicking region in Sen1

Pervasive transcription is a widespread phenomenon leading to the production of a plethora of non-coding RNAs (ncRNAs) without apparent function. Pervasive transcription poses a threat to proper gene expression that needs to be controlled. In yeast, the highly conserved helicase Sen1 restricts pervasive transcription by inducing termination of non-coding transcription. However, the mechanisms underlying the specific function of Sen1 at ncRNAs are poorly understood. Here, we identify a motif in an intrinsically disordered region of Sen1 that mimics the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II, and structurally characterize its recognition by the CTD-interacting domain of Nrd1, an RNA-binding protein that binds specific sequences in ncRNAs. In addition, we show that Sen1-dependent termination strictly requires CTD recognition by the N-terminal domain of Sen1. We provide evidence that the Sen1-CTD interaction does not promote initial Sen1 recruitment, but rather enhances Sen1 capacity to induce the release of paused RNAPII from the DNA. Our results shed light on the network of protein-protein interactions that control termination of non-coding transcription by Sen1

High-sensitive nascent transcript sequencing reveals BRD4-specific control of widespread enhancer and target gene transcription

Gene transcription by RNA polymerase II (Pol II) is under control of promoters and distal regulatory elements known as enhancers. Enhancers are themselves transcribed by Pol II correlating with their activity. How enhancer transcription is regulated and coordinated with transcription at target genes has remained unclear. Here, we developed a high-sensitive native elongating transcript sequencing approach, called HiS-NET-seq, to provide an extended high-resolution view on transcription, especially at lowly transcribed regions such as enhancers. HiS-NET-seq uncovers new transcribed enhancers in human cells. A multi-omics analysis shows that genome-wide enhancer transcription depends on the BET family protein BRD4. Specifically, BRD4 co-localizes to enhancer and promoter-proximal gene regions, and is required for elongation activation at enhancers and their genes. BRD4 keeps a set of enhancers and genes in proximity through long-range contacts. From these studies BRD4 emerges as a general regulator of enhancer transcription that may link transcription at enhancers and genes

C-Terminal Extension of the Yeast Mitochondrial DNA Polymerase Determines the Balance between Synthesis and Degradation

Saccharomyces cerevisiae mitochondrial DNA polymerase (Mip1) contains a C-terminal extension (CTE) of 279 amino acid residues. The CTE is required for mitochondrial DNA maintenance in yeast but is absent in higher eukaryotes. Here we use recombinant Mip1 C-terminal deletion mutants to investigate functional importance of the CTE. We show that partial removal of the CTE in Mip1Δ216 results in strong preference for exonucleolytic degradation rather than DNA polymerization. This disbalance in exonuclease and polymerase activities is prominent at suboptimal dNTP concentrations and in the absence of correctly pairing nucleotide. Mip1Δ216 also displays reduced ability to synthesize DNA through double-stranded regions. Full removal of the CTE in Mip1Δ279 results in complete loss of Mip1 polymerase activity, however the mutant retains its exonuclease activity. These results allow us to propose that CTE functions as a part of Mip1 polymerase domain that stabilizes the substrate primer end at the polymerase active site, and is therefore required for efficient mitochondrial DNA replication in vivo

Conserved DNA sequence features underlie pervasive RNA polymerase pausing

Pausing of transcribing RNA polymerase is regulated and creates opportunities to control gene expression. Research in metazoans has so far mainly focused on RNA polymerase II (Pol II) promoter-proximal pausing leaving the pervasive nature of pausing and its regulatory potential in mammalian cells unclear. Here, we developed a pause detecting algorithm (PDA) for nucleotide-resolution occupancy data and a new native elongating transcript sequencing approach, termed nested NET-seq, that strongly reduces artifactual peaks commonly misinterpreted as pausing sites. Leveraging PDA and nested NET-seq reveal widespread genome-wide Pol II pausing at single-nucleotide resolution in human cells. Notably, the majority of Pol II pauses occur outside of promoter-proximal gene regions primarily along the gene-body of transcribed genes. Sequence analysis combined with machine learning modeling reveals DNA sequence properties underlying widespread transcriptional pausing including a new pause motif. Interestingly, key sequence determinants of RNA polymerase pausing are conserved between human cells and bacteria. These studies indicate pervasive sequence-induced transcriptional pausing in human cells and the knowledge of exact pause locations implies potential functional roles in gene expression

Exonuclease activity of Mip1 and C-terminal deletion mutants.

<p>Exonuclease activity was assayed on a 45 nt template primed with a radiolabeled 25 nt primer. 4 nM DNA polymerase was incubated with 2 nM 45/25 substrate in the total absence of dNTP or in the presence of 5 nM dATP. Reactions were carried out at 30°C and stopped at indicated time points with equal volume of 80% formamide, 25 mM EDTA. A. Reaction products without dNTP were resolved on 8% urea polyacrylamide gel. B. Reaction products with 5 nM dATP were resolved on 8% urea polyacrylamide gel. Position of the 25 nt primer is indicated. C. Exonuclease activity was calculated as the amount of released dNMP from the reaction products. The proportional amount of the products was calculated from the intensity of the signal and the amount of the dNMP released during the reaction, taking into account the size of each of the exonuclease products. The amount of released dNMP was plotted against time. Filled square – reaction in the absence of the dNTP, empty circle – reaction in the presence of 5 nM dATP.</p

Polymerase/exonuclease balance of Mip1 and C-terminal deletion mutants.

<p>The balance between polymerase and exonuclease activities was assayed on a 45 nt template primed with a radiolabeled 25 nt primer. 4 nM DNA polymerase and 10 nM substrate were incubated for 5 min at 30°C in the presence of indicated concentrations of dNTP. Reactions were stopped with an equal volume of 80% formamide, 25 mM EDTA. A. Reaction products were resolved on 8% urea polyacrylamide gel. Positions of the 25 nt primer and 45 nt reaction product are indicated. B. The percentage of the polymerization products out of total reaction products were plotted against dNTP concentration. FL-Mip1 – empty circle, Mip1Δ175 –filled square, Mip1Δ216 – filled triangle.</p

Processivity of Mip1 and C-terminal deletion mutants.

<p>Processivity was measured under single-hit conditions with 4 nM of substrate M13 ssDNA singly primed with radiolabeled USP and 1 mg/ml calf thymus activated DNA. The reaction was performed at 30°C with 4 nM DNA polymerase in the presence of 100 µM dNTP. The reaction was stopped with 0.5 mg/ml Proteinase K, 1% SDS, 20 mM EDTA after indicated time points. A. Reaction products were separated on 0.8% alkaline agarose gel. Arrows indicate positions of M13mp18 unit length (7250 nt) and 17 nt USP. B. Processivity of FL-Mip1, Mip1Δ175 and Mip1Δ216 was calculated as the average length of the product (nt) synthesized by the polymerase per one binding event. Weighted mean method based on the product intensity and length was used for analysis. Data from three independent experiments was used to calculate the average processivity and standard deviation values.</p