Finn Werner

The Initiation Factor TFE and the Elongation Factor Spt4/5 Compete for the RNAP Clamp during Transcription Initiation and Elongation

Summary TFIIE and the archaeal homolog TFE enhance DNA strand separation of eukaryotic RNAPII and the archaeal RNAP during transcription initiation by an unknown mechanism. We have developed a fluorescently labeled recombinant M. jannaschii RNAP system to probe the archaeal transcription initiation complex, consisting of promoter DNA, TBP, TFB, TFE, and RNAP. We have localized the position of the TFE winged helix (WH) and Zinc ribbon (ZR) domains on the RNAP using single-molecule FRET. The interaction sites of the TFE WH domain and the transcription elongation factor Spt4/5 overlap, and both factors compete for RNAP binding. Binding of Spt4/5 to RNAP represses promoter-directed transcription in the absence of TFE, which alleviates this effect by displacing Spt4/5 from RNAP. During elongation, Spt4/5 can displace TFE from the RNAP elongation complex and stimulate processivity. Our results identify the RNAP “clamp” region as a regulatory hot spot for both transcription initiation and transcription elongation.

Direct modulation of RNA polymerase core functions by basal transcription factors

ABSTRACT Archaeal RNA polymerases (RNAPs) are recruited to promoters through the joint action of three basal transcription factors: TATA-binding protein, TFB (archaeal homolog of TFIIB), and TFE (archaeal homolog of TFIIE). Our results demonstrate several new insights into the mechanisms of TFB and TFE during the transcription cycle. (i) The N-terminal Zn ribbon of TFB displays a surprising degree of redundancy for the recruitment of RNAP during transcription initiation in the archaeal system. (ii) The B-finger domain of TFB participates in transcription initiation events by stimulating abortive and productive transcription in a recruitment-independent function. TFB thus combines physical recruitment of the RNAP with an active role in influencing the catalytic properties of RNAP during transcription initiation. (iii) TFB mutations are complemented by TFE, thereby demonstrating that both factors act synergistically during transcription initiation. (iv) An additional function of TFE is to dynamically alter the nucleic acid-binding properties of RNAP by stabilizing the initiation complex and destabilizing elongation complexes.

Tunable reagents for multi-functional bioconjugation: reversible or permanent chemical modification of proteins and peptides by control of maleimide hydrolysis

Controlling maleimide hydrolysis allows the modular construction of bromomaleimide-mediated bioconjugates which are either stable or cleavable in an aqueous, thiol-mediated reducing environment.

A Nexus for Gene Expression—Molecular Mechanisms of Spt5 and NusG in the Three Domains of Life

Evolutionary related multisubunit RNA polymerases (RNAPs) transcribe the genomes of all living organisms. Whereas the core subunits of RNAPs are universally conserved in all three domains of life—indicative of a common evolutionary descent—this only applies to one RNAP-associated transcription factor—Spt5, also known as NusG in bacteria. All other factors that aid RNAP during the transcription cycle are specific for the individual domain or only conserved between archaea and eukaryotes. Spt5 and its bacterial homologue NusG regulate gene expression in several ways by (i) modulating transcription processivity and promoter proximal pausing, (ii) coupling transcription and RNA processing or translation, and (iii) recruiting termination factors and thereby silencing laterally transferred DNA and protecting the genome against double-stranded DNA breaks. This review discusses recent discoveries that identify Spt5-like factors as evolutionary conserved nexus for the regulation and coordination of the machineries responsible for information processing in the cell.

Formation and Abundance of 5-Hydroxymethylcytosine in RNA

RNA methylation is emerging as a regulatory RNA modification that could have important roles in the control and coordination of gene transcription and protein translation. Herein, we describe an in vivo isotope‐tracing methodology to demonstrate that the ribonucleoside 5‐methylcytidine (m5C) is subject to oxidative processing in mammals, forming 5‐hydroxymethylcytidine (hm5C) and 5‐formylcytidine (f5C). Furthermore, we have identified hm5C in total RNA from all three domains of life and in polyA‐enriched RNA fractions from mammalian cells. This suggests m5C oxidation is a conserved process that could have critical regulatory functions inside cells.

Molecular mechanisms of RNA polymerase—the F/E (RPB4/7) complex is required for high processivity in vitro

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.

Simulation vs. reality: a comparison of in silico distance predictions with DEER and FRET measurements.

Site specific incorporation of molecular probes such as fluorescent- and nitroxide spin-labels into biomolecules, and subsequent analysis by Förster resonance energy transfer (FRET) and double electron-electron resonance (DEER) can elucidate the distance and distance-changes between the probes. However, the probes have an intrinsic conformational flexibility due to the linker by which they are conjugated to the biomolecule. This property minimizes the influence of the label side chain on the structure of the target molecule, but complicates the direct correlation of the experimental inter-label distances with the macromolecular structure or changes thereof. Simulation methods that account for the conformational flexibility and orientation of the probe(s) can be helpful in overcoming this problem. We performed distance measurements using FRET and DEER and explored different simulation techniques to predict inter-label distances using the Rpo4/7 stalk module of the M. jannaschii RNA polymerase. This is a suitable model system because it is rigid and a high-resolution X-ray structure is available. The conformations of the fluorescent labels and nitroxide spin labels on Rpo4/7 were modeled using in vacuo molecular dynamics simulations (MD) and a stochastic Monte Carlo sampling approach. For the nitroxide probes we also performed MD simulations with explicit water and carried out a rotamer library analysis. Our results show that the Monte Carlo simulations are in better agreement with experiments than the MD simulations and the rotamer library approach results in plausible distance predictions. Because the latter is the least computationally demanding of the methods we have explored, and is readily available to many researchers, it prevails as the method of choice for the interpretation of DEER distance distributions.

Recent advances in the understanding of archaeal transcription.

RNA polymerases (RNAPs) make repeatedly use of their templates by cycling through initiation, elongation and termination phases of transcription; during each step RNAP is interacting with and regulated by distinct transcription factors. The dynamic interplay between nucleic acid sequences, transcription factors and RNAP affects the activity and distribution of transcription complexes across the genome, and ultimately executes the genetic programme of the organism. This review covers recent discoveries about the mechanisms of archaeal transcription obtained by a combination of in vivo and in vitro approaches, from the molecular to the global level.

The interplay between nucleoid organization and transcription in archaeal genomes

The archaeal genome is organized by either eukaryotic-like histone proteins or bacterial-like nucleoid-associated proteins. Recent studies have revealed novel insights into chromatin dynamics and their effect on gene expression in archaeal model organisms. In this Progress article, we discuss the interplay between chromatin proteins, such as histones and Alba, and components of the basal transcription machinery, as well as between chromatin structure and gene-specific transcription factors in archaea. Such an interplay suggests that chromatin might have a role in regulating gene expression on both a global and a gene-specific level. Moreover, several archaeal transcription factors combine a global gene regulatory role with an architectural role, thus contributing to chromatin organization and compaction, as well as gene expression. We describe the emerging principles underlying how these factors cooperate in nucleoid structuring and gene regulation.

Complete architecture of the archaeal RNA polymerase open complex from single-molecule FRET and NPS.

The molecular architecture of RNAP II-like transcription initiation complexes remains opaque due to its conformational flexibility and size. Here we report the three-dimensional architecture of the complete open complex (OC) composed of the promoter DNA, TATA box-binding protein (TBP), transcription factor B (TFB), transcription factor E (TFE) and the 12-subunit RNA polymerase (RNAP) from Methanocaldococcus jannaschii. By combining single-molecule Förster resonance energy transfer and the Bayesian parameter estimation-based Nano-Positioning System analysis, we model the entire archaeal OC, which elucidates the path of the non-template DNA (ntDNA) strand and interaction sites of the transcription factors with the RNAP. Compared with models of the eukaryotic OC, the TATA DNA region with TBP and TFB is positioned closer to the surface of the RNAP, likely providing the mechanism by which DNA melting can occur in a minimal factor configuration, without the dedicated translocase/helicase encoding factor TFIIH.