NDF is a transcription factor that stimulates elongation by RNA polymerase II.

RNA polymerase II (Pol II) elongation is a critical step in gene expression. Here we found that NDF, which was identified as a bilaterian nucleosome-destabilizing factor, is also a Pol II transcription factor that stimulates elongation with plain DNA templates in the absence of nucleosomes. NDF binds directly to Pol II and enhances elongation by a different mechanism than that used by transcription factor TFIIS.

The tardigrade damage suppressor protein binds to nucleosomes and protects DNA from hydroxyl radicals.

Tardigrades, also known as water bears, are animals that can survive extreme conditions. The tardigrade contains a unique nuclear protein termed Dsup, for damage suppressor, which can increase the resistance of human cells to DNA damage under conditions, such as ionizing radiation or hydrogen peroxide treatment, that generate hydroxyl radicals. Here we find that Dsup is a nucleosome-binding protein that protects chromatin from hydroxyl radicals.

The RNA Polymerase II Core Promoter in .

Transcription by RNA polymerase II initiates at the core promoter, which is sometimes referred to as the "gateway to transcription." Here, we describe the properties of the RNA polymerase II core promoter in The core promoter is at a strategic position in the expression of genes, as it is the site of convergence of the signals that lead to transcriptional activation. Importantly, core promoters are diverse in terms of their structure and function.

Molecular basis of chromatin remodeling by Rhp26, a yeast CSB ortholog.

CSB/ERCC6 belongs to an orphan subfamily of SWI2/SNF2-related chromatin remodelers and plays crucial roles in gene expression, DNA damage repair, and the maintenance of genome integrity. The molecular basis of chromatin remodeling by Cockayne syndrome B protein (CSB) is not well understood. Here we investigate the molecular mechanism of chromatin remodeling by Rhp26, a CSB ortholog.

NDF, a nucleosome-destabilizing factor that facilitates transcription through nucleosomes.

Our understanding of transcription by RNA polymerase II (Pol II) is limited by our knowledge of the factors that mediate this critically important process. Here we describe the identification of NDF, a nucleosome-destabilizing factor that facilitates Pol II transcription in chromatin. NDF has a PWWP motif, interacts with nucleosomes near the dyad, destabilizes nucleosomes in an ATP-independent manner, and facilitates transcription by Pol II through nucleosomes in a purified and defined transcription system as well as in cell nuclei.

The punctilious RNA polymerase II core promoter.

The signals that direct the initiation of transcription ultimately converge at the core promoter, which is the gateway to transcription. Here we provide an overview of the RNA polymerase II core promoter in bilateria (bilaterally symmetric animals). The core promoter is diverse in terms of its composition and function yet is also punctilious, as it acts with strict rules and precision.

The prenucleosome, a stable conformational isomer of the nucleosome.

Chromatin comprises nucleosomes as well as nonnucleosomal histone-DNA particles. Prenucleosomes are rapidly formed histone-DNA particles that can be converted into canonical nucleosomes by a motor protein such as ACF. Here we show that the prenucleosome is a stable conformational isomer of the nucleosome.

Transcription. Comment on "Mechanism of eukaryotic RNA polymerase III transcription termination".

Nielsen et al. (Reports, 28 June 2013, p. 1577) characterized their RNA polymerase III (Pol III) preparation and concluded that it requires an RNA hairpin/duplex structure for terminating transcription.

TRF2, but not TBP, mediates the transcription of ribosomal protein genes.

The TCT core promoter element is present in most ribosomal protein (RP) genes in Drosophila and humans. Here we show that TBP (TATA box-binding protein)-related factor TRF2, but not TBP, is required for transcription of the TCT-dependent RP genes. In cells, TCT-dependent transcription, but not TATA-dependent transcription, increases or decreases upon overexpression or depletion of TRF2.

The annealing helicase and branch migration activities of Drosophila HARP.

HARP (SMARCAL1, MARCAL1) is an annealing helicase that functions in the repair and restart of damaged DNA replication forks through its DNA branch migration and replication fork regression activities. HARP is conserved among metazoans. HARP from invertebrates differs by the absence of one of the two HARP-specific domain repeats found in vertebrates.

Human TFIID binds to core promoter DNA in a reorganized structural state.

A mechanistic description of metazoan transcription is essential for understanding the molecular processes that govern cellular decisions. To provide structural insights into the DNA recognition step of transcription initiation, we used single-particle electron microscopy (EM) to visualize human TFIID with promoter DNA. This analysis revealed that TFIID coexists in two predominant and distinct structural states that differ by a 100 Å translocation of TFIID's lobe A.

Transcription of the T4 late genes.

This article reviews the current state of understanding of the regulated transcription of the bacteriophage T4 late genes, with a focus on the underlying biochemical mechanisms, which turn out to be unique to the T4-related family of phages or significantly different from other bacterial systems. The activator of T4 late transcription is the gene 45 protein (gp45), the sliding clamp of the T4 replisome. Gp45 becomes topologically linked to DNA through the action of its clamp-loader, but it is not site-specifically DNA-bound, as other transcriptional activators are.

Molecular basis of RNA polymerase III transcription repression by Maf1.

RNA polymerase III (Pol III) transcribes short RNAs required for cell growth. Under stress conditions, the conserved protein Maf1 rapidly represses Pol III transcription. We report the crystal structure of Maf1 and cryo-electron microscopic structures of Pol III, an active Pol III-DNA-RNA complex, and a repressive Pol III-Maf1 complex.

The C53/C37 subcomplex of RNA polymerase III lies near the active site and participates in promoter opening.

The C53 and C37 subunits of RNA polymerase III (pol III) form a subassembly that is required for efficient termination; pol III lacking this subcomplex displays increased processivity of RNA chain elongation. We show that the C53/C37 subcomplex additionally plays a role in formation of the initiation-ready open promoter complex similar to that of the Brf1 N-terminal zinc ribbon domain. In the absence of C53 and C37, the transcription bubble fails to stably propagate to and beyond the transcriptional start site even when the DNA template is supercoiled.

Absolute gene occupancies by RNA polymerase III, TFIIIB, and TFIIIC in Saccharomyces cerevisiae.

A major limitation of chromatin immunoprecipitation lies in the challenge of measuring the immunoprecipitation effectiveness of different proteins and antibodies and the resultant inability to compare the occupancies of different DNA-binding proteins. Here we present the implementation of a quantitative chromatin immunoprecipitation assay in the RNA polymerase III (pol III) system that allowed us to measure the absolute in vivo occupancy of pol III and its two transcription factors, TFIIIC and TFIIIB, on a subset of pol III genes. The crucial point of our analysis was devising a method that allows the accurate determination of the immunoprecipitation efficiency for each protein.

Transcription: adjusting to adversity by regulating RNA polymerase.

Under growth-limiting conditions, budding yeast shut down transcription of genes of the translation apparatus. Recent studies have shown that this response is signaled, in part, by multiple pathways that converge on Maf1, leading to a change of this protein's phosphorylation state and its relocation to the nucleus, where it represses RNA polymerase III.

Mapping the principal interaction site of the Brf1 and Bdp1 subunits of Saccharomyces cerevisiae TFIIIB.

The Brf1 subunit of the central RNA polymerase (pol) III transcription initiation factor TFIIIB is bipartite; its N-terminal TFIIB-related half is principally responsible for recruiting pol III to the promoter and for promoter opening near the transcriptional start site, whereas its pol III-specific C-terminal half contributes most of the affinities that hold the three subunits of TFIIIB together. Here, the principal attachment site of Brf1 for the Bdp1 subunit of TFIIIB has been mapped by a combination of structure-informed, site-directed mutagenesis and photochemical protein-DNA cross-linking. A 66-amino acid segment of Brf1 is shown to serve as a two-sided adhesive surface, with the side chains projecting away from its extended interface with TATA-binding protein anchoring Bdp1 binding.

Nhp6 is a transcriptional initiation fidelity factor for RNA polymerase III transcription in vitro and in vivo.

The binding of the RNA polymerase III (pol III) transcription factor TFIIIC to the box A intragenic promoter element of tRNA genes specifies the placement of TFIIIB on upstream-lying DNA. In turn, TFIIIB recruits pol III to the promoter and specifies transcription initiating 17-19 base pairs upstream of box A. The resolution of the pol III transcription apparatus into recombinant TFIIIB, highly purified TFIIIC, and pol III is accompanied by a loss of precision in specifying where transcription initiation occurs due to heterogeneous placement of TFIIIB.

Reconfiguring the connectivity of a multiprotein complex: fusions of yeast TATA-binding protein with Brf1, and the function of transcription factor IIIB.

Transcription factor (TF) IIIB, the central transcription initiation factor of RNA polymerase III (pol III), is composed of three subunits, Bdp1, Brf1 and TATA-binding protein (TBP), all essential for normal function in vivo and in vitro. Brf1 is a modular protein: Its N-proximal half is related to TFIIB and binds similarly to the C-terminal stirrup of TBP; its C-proximal one-third provides most of the affinity for TBP by binding along the entire length of the convex surface and N-terminal lateral face of TBP. A structure-informed triple fusion protein, with TBP core placed between the N- and C-proximal domains of Brf1, has been constructed.

High-level activation of transcription of the yeast U6 snRNA gene in chromatin by the basal RNA polymerase III transcription factor TFIIIC.

Transcription of the U6 snRNA gene (SNR6) in Saccharomyces cerevisiae by RNA polymerase III (pol III) requires TFIIIC and its box A and B binding sites. In contrast, TFIIIC has little or no effect on SNR6 transcription with purified components in vitro due to direct recognition of the SNR6 TATA box by TFIIIB. When SNR6 was assembled into chromatin in vitro by use of the Drosophila melanogaster S-190 extract, transcription of these templates with highly purified yeast pol III, TFIIIC, and TFIIIB displayed a near-absolute requirement for TFIIIC but yielded a 5- to 15-fold-higher level of transcription relative to naked DNA (>100-fold activation over repressed chromatin).

A common site on TBP for transcription by RNA polymerases II and III.

The TATA-binding protein (TBP) is involved in all nuclear transcription. We show that a common site on TBP is used for transcription initiation complex formation by RNA polymerases (pols) II and III. TBP, the transcription factor IIB (TFIIB)-related factor Brf1 and the pol III-specific factor Bdp1 constitute TFIIIB.

A gene-specific effect of an internal deletion in the Bdp1 subunit of the RNA polymerase III transcription initiation factor TFIIIB.

The Saccharomyces cerevisiae RPR1 gene encodes the RNA subunit of its RNase P, which processes RNA polymerase (pol) III primary transcripts. RPR1, which is transcribed by pol III, has been isolated as a multicopy suppressor of a specific small internal deletion (amino acids 253-269) in the Bdp1 subunit of transcription factor TFIIIB, the core pol III transcription factor. The selective effect of this Bdp1 deletion on RPR1 transcription has been analyzed in vitro.

Crystal structure of a transcription factor IIIB core interface ternary complex.

Transcription factor IIIB (TFIIIB), consisting of the TATA-binding protein (TBP), TFIIB-related factor (Brf1) and Bdp1, is a central component in basal and regulated transcription by RNA polymerase III. TFIIIB recruits its polymerase to the promoter and subsequently has an essential role in the formation of the open initiation complex. The amino-terminal half of Brf1 shares a high degree of sequence similarity with the polymerase II general transcription factor TFIIB, but it is the carboxy-terminal half of Brf1 that contributes most of its binding affinity with TBP.

The role of transcription initiation factor IIIB subunits in promoter opening probed by photochemical cross-linking.

The core transcription initiation factor (TF) IIIB recruits its conjugate RNA polymerase (pol) III to the promoter and also plays an essential role in promoter opening. TFIIIB assembled with certain deletion mutants of its Brf1 and Bdp1 subunits is competent in pol III recruitment, but the resulting preinitiation complex does not open the promoter. Whether Brf1 and Bdp1 participate in opening the promoter by direct DNA interaction (as sigma subunits of bacterial RNA polymerases do) or indirectly by their action on pol III has been approached by site-specific photochemical protein-DNA cross-linking of TFIIIB-pol III-U6 RNA gene promoter complexes.

A single-stranded promoter for RNA polymerase III.

Single strands of DNA serve, in rare instances, as promoters for transcription; duplex DNA promoters with individual strands that also have a promoter capacityfunction have not been described. We show that the nontranscribed strand of the Saccharomyces cerevisiae U6 snRNA gene directs transcription initiation factor IIIB-requiring and accurately initiating transcription by RNA polymerase III. The nontranscribed strand promoter is much more extended than its duplex DNA counterpart, comprising the U6 gene TATA box, a downstream T(7) tract, and an upstream-lying segment.