Quantitative insights into processivity of an Hsp100 protein disaggregase on folded proteins.

The Hsp100 family of protein disaggregases plays important roles in maintaining protein homeostasis in cells. E. coli ClpB is an Hsp100 protein that solubilizes protein aggregates.

Single turnover transient state kinetics reveals processive protein unfolding catalyzed by ClpB.

ClpB and Hsp104 are AAA+ motor proteins essential for proteome maintenance and thermal tolerance. ClpB and Hsp104 have been proposed to extract a polypeptide from an aggregate and processively translocate the chain through the axial channel of its hexameric ring structure. However, the mechanism of translocation and if this reaction is processive remains disputed.

Biochemical characterization of RNA polymerases.

Tuberculosis is caused by the bacterium (Mtb). While eukaryotic species employ several specialized RNA polymerases (Pols) to fulfill the RNA synthesis requirements of the cell, bacterial species use a single RNA polymerase (RNAP). To contribute to the foundational understanding of how Mtb and the related non-pathogenic mycobacterial species, (Msm), perform the essential function of RNA synthesis, we performed a series of transcription experiments to define the unique enzymatic properties of Mtb and Msm RNAPs.

NTPs compete in the active site of RNA polymerases I and II.

Eukaryotes express at least three RNA polymerases (Pols) carry out transcription, while bacteria and archaea use only one. Using transient state kinetics, we have extensively examined and compared the kinetics of both single and multi-nucleotide additions catalyzed by the three Pols. In single nucleotide addition experiments we have observed unexpected extension products beyond one incorporation, which can be attributed to misincorporation, the presence of nearly undetectable amounts of contaminating NTPs, or a mixture of the two.

Global kinetic mechanism describing single nucleotide incorporation for RNA polymerase I reveals fast UMP incorporation.

RNA polymerase I (Pol I) is responsible for synthesizing ribosomal RNA, which is the rate limiting step in ribosome biogenesis. We have reported wide variability in the magnitude of the rate constants defining the rate limiting step in sequential nucleotide additions catalyzed by Pol I. in this study we sought to determine if base identity impacts the rate limiting step of nucleotide addition catalyzed by Pol I.

Reversible Kinetics in Multi-nucleotide Addition Catalyzed by S. cerevisiae RNA polymerase II Reveal Slow Pyrophosphate Release.

Eukaryotes express at least three nuclear DNA dependent RNA polymerases (Pols). Pols I, II, and III synthesize ribosomal (r) RNA, messenger (m) RNA, and transfer (t) RNA, respectively. Pol I and Pol III have intrinsic nuclease activity conferred by the A12.

Quantifying the impact of initial RNA primer length on nucleotide addition by RNA polymerase I.

Transient state kinetic studies of eukaryotic DNA-dependent RNA polymerases (Pols) in vitro provide quantitative characterization of enzyme activity at the level of individual nucleotide addition events. Previous work revealed heterogeneity in the rate constants governing nucleotide addition by yeast RNA polymerase I (Pol I) for each position on a template DNA. In contrast, the rate constants that described nucleotide addition by yeast RNA polymerase II (Pol II) were more homogeneous.

The A12.2 Subunit Plays an Integral Role in Pyrophosphate Release of RNA Polymerase I.

RNA polymerase I (Pol I) synthesizes ribosomal RNA (rRNA), which is the first and rate-limiting step in ribosome biosynthesis. A12.2 (A12) is a critical subunit of Pol I that is responsible for activating Pol I's exonuclease activity.

Protocol for monitoring and analyzing single nucleotide incorporation by S. cerevisiae RNA polymerases.

Here we present an optimized protocol for monitoring and analyzing single nucleotide incorporation by RNA polymerases. This protocol describes the assembly of Saccharomyces cerevisiae RNA polymerase I elongation complexes in a promoter-independent system in vitro. We describe how to collect a time course using a quench-flow, a rapid mixing instrument, and subsequently resolve reactions on a polyacrylamide gel.

The cofactor-dependent folding mechanism of Drosophila cryptochrome revealed by single-molecule pulling experiments.

The link between cofactor binding and protein activity is well-established. However, how cofactor interactions modulate folding of large proteins remains unknown. We use optical tweezers, clustering and global fitting to dissect the folding mechanism of Drosophila cryptochrome (dCRY), a 542-residue protein that binds FAD, one of the most chemically and structurally complex cofactors in nature.

Transient-State Kinetic Analysis of the RNA Polymerase II Nucleotide Incorporation Mechanism.

Eukaryotic RNA polymerase II (Pol II) is an essential enzyme that lies at the core of eukaryotic biology. Due to its pivotal role in gene expression, Pol II has been subjected to a substantial number of investigations. We aim to further our understanding of Pol II nucleotide incorporation by utilizing transient-state kinetic techniques to examine Pol II single nucleotide addition on the millisecond time scale.

RNA Polymerase I Is Uniquely Vulnerable to the Small-Molecule Inhibitor BMH-21.

Cancer cells require robust ribosome biogenesis to maintain rapid cell growth during tumorigenesis. Because RNA polymerase I (Pol I) transcription of the ribosomal DNA (rDNA) is the first and rate-limiting step of ribosome biogenesis, it has emerged as a promising anti-cancer target. Over the last decade, novel cancer therapeutics targeting Pol I have progressed to clinical trials.

Uncovering the mechanisms of transcription elongation by eukaryotic RNA polymerases I, II, and III.

Eukaryotes express three nuclear RNA polymerases (Pols I, II, and III) that are essential for cell survival. Despite extensive investigation of the three Pols, significant knowledge gaps regarding their biochemical properties remain because each Pol has been evaluated independently under disparate experimental conditions and methodologies. To advance our understanding of the Pols, we employed identical transcription assays for direct comparison of their elongation rates, elongation complex (EC) stabilities, and fidelities.

AAA+ proteins: one motor, multiple ways to work.

Numerous ATPases associated with diverse cellular activities (AAA+) proteins form hexameric, ring-shaped complexes that function via ATPase-coupled translocation of substrates across the central channel. Cryo-electron microscopy of AAA+ proteins processing substrate has revealed non-symmetric, staircase-like hexameric structures that indicate a sequential clockwise/2-residue step translocation model for these motors. However, for many of the AAA+ proteins that share similar structural features, their translocation properties have not yet been experimentally determined.

Multi-start Evolutionary Nonlinear OpTimizeR (MENOTR): A hybrid parameter optimization toolbox.

Parameter optimization or "data fitting" is a computational process that identifies a set of parameter values that best describe an experimental data set. Parameter optimization is commonly carried out using a computer program utilizing a non-linear least squares (NLLS) algorithm. These algorithms work by continuously refining a user supplied initial guess resulting in a systematic increase in the goodness of fit.

Transient-state kinetic analysis of multi-nucleotide addition catalyzed by RNA polymerase I.

RNA polymerases execute the first step in gene expression: transcription of DNA into RNA. Eukaryotes, unlike prokaryotes, express at least three specialized nuclear multisubunit RNA polymerases (Pol I, Pol II, and Pol III). RNA polymerase I (Pol I) synthesizes the most abundant RNA, ribosomal RNA.

The N-terminal domain of the A12.2 subunit stimulates RNA polymerase I transcription elongation.

Eukaryotes express three DNA-dependent RNA polymerases (Pols) that are responsible for the entirety of cellular genomic expression. The three Pols have evolved to express specific cohorts of RNAs and thus have diverged both structurally and functionally to efficiently execute their specific transcriptional roles. One example of this divergence is Pol I's inclusion of a proofreading factor as a bona fide subunit, as opposed to Pol II, which recruits a transcription factor, TFIIS, for proofreading.

Defining the divergent enzymatic properties of RNA polymerases I and II.

Eukaryotes express at least three nuclear DNA-dependent RNA polymerases (Pols) responsible for synthesizing all RNA required by the cell. Despite sharing structural homology, they have functionally diverged to suit their distinct cellular roles. Although the Pols have been studied extensively, direct comparison of their enzymatic properties is difficult because studies are often conducted under disparate experimental conditions and techniques.

Kinetic Analysis of AAA+ Translocases by Combined Fluorescence and Anisotropy Methods.

The multitude of varied, energy-dependent processes that exist in the cell necessitate a diverse array of macromolecular machines to maintain homeostasis, allow for growth, and facilitate reproduction. ATPases associated with various cellular activity are a set of protein assemblies that function as molecular motors to couple the energy of nucleoside triphosphate binding and hydrolysis to mechanical movement along a polymer lattice. A recent boom in structural insights into these motors has led to structural hypotheses on how these motors fulfill their function.

Conformational plasticity of the ClpAP AAA+ protease couples protein unfolding and proteolysis.

The ClpAP complex is a conserved bacterial protease that unfolds and degrades proteins targeted for destruction. The ClpA double-ring hexamer powers substrate unfolding and translocation into the ClpP proteolytic chamber. Here, we determined high-resolution structures of wild-type Escherichia coli ClpAP undergoing active substrate unfolding and proteolysis.

Downstream sequence-dependent RNA cleavage and pausing by RNA polymerase I.

The sequence of the DNA template has long been thought to influence the rate of transcription by DNA-dependent RNA polymerases, but the influence of DNA sequence on transcription elongation properties of eukaryotic RNA polymerase I (Pol I) from has not been defined. In this study, we observe changes in dinucleotide production, transcription elongation complex stability, and Pol I pausing in response to downstream DNA. studies demonstrate that AT-rich downstream DNA enhances pausing by Pol I and inhibits Pol I nucleolytic cleavage activity.

Examination of the nucleotide-linked assembly mechanism of E. coli ClpA.

Escherichia coli ClpA is a AAA+ (ATPase Associated with diverse cellular Activities) chaperone that catalyzes the ATP-dependent unfolding and translocation of substrate proteins targeted for degradation by a protease, ClpP. ClpA hexamers associate with one or both ends of ClpP tetradecamers to form ClpAP complexes. Each ClpA protomer contains two nucleotide-binding sites, NBD1 and NBD2, and self-assembly into hexamers is thermodynamically linked to nucleotide binding.

Hsp104 and Potentiated Variants Can Operate as Distinct Nonprocessive Translocases.

Heat shock protein (Hsp) 104 is a hexameric ATPases associated with diverse cellular activities motor protein that enables cells to survive extreme stress. Hsp104 couples the energy of ATP binding and hydrolysis to solubilize proteins trapped in aggregated structures. The mechanism by which Hsp104 disaggregates proteins is not completely understood but may require Hsp104 to partially or completely translocate polypeptides across its central channel.

A Novel Assay for RNA Polymerase I Transcription Elongation Sheds Light on the Evolutionary Divergence of Eukaryotic RNA Polymerases.

Eukaryotic cells express at least three nuclear RNA polymerases (Pols), each with a unique set of gene targets. Though these enzymes are homologous, there are many differences among the Pols. In this study, a novel assay for Pol I transcription elongation was developed to probe enzymatic differences among the Pols.

ATP hydrolysis inactivating Walker B mutation perturbs E. coli ClpA self-assembly energetics in the absence of nucleotide.

E. coli ClpA is an AAA+ (ATPase Associated with diverse cellular Activities) chaperone that catalyzes the ATP-dependent unfolding and translocation of substrate proteins for the purposes of proper proteome maintenance. Biologically active ClpA hexamers contain two nucleotide binding domains (NBD) per protomer, D1 and D2.