Springer protocols feed by Microbiology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Site-Specific Incorporation of Probes into RNA Polymerase by Unnatural-Amino-Acid Mutagenesis and Staudinger–Bertozzi Ligation
We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - February 13, 2015 Category: Microbiology Source Type: news
Methods for the Assembly and Analysis of In Vitro Transcription-Coupled-to-Translation Systems
RNA polymerase is a complex machinery, which is further embedded in interactions with other cellular components that interplay with either the transcribed DNA (DNA polymerases, topoisomerases, etc.) or the nascent RNA (RNA processing enzymes, ribosomes, etc.). In prokaryotes, coupling of transcription and translation is thought to play many regulatory roles but the mechanistic understanding of their interactions has been hindered by the lack of a defined experimental system. Here, we describe a pure transcription-coupled-to-translation system in which control of the ribosome has been achieved through its stepwise transloca...
Source: Springer protocols feed by Microbiology - February 13, 2015 Category: Microbiology Source Type: news
In Vitro and In Vivo Methodologies for Studying the Sigma 54-Dependent Transcription
Here we describe approaches and methods to assaying in vitro the major variant bacterial sigma factor, Sigma 54 (σ54), in a purified system. We include the complete transcription system, binding interactions between σ54 and its activators, as well as the self-assembly and the critical ATPase activity of the cognate activators which serve to remodel the closed promoter complexes. We also present in vivo methodologies that are used to study the impact of physiological processes, metabolic states, global signalling networks, and cellular architecture on the control of σ54-dependent gene expression. (Source: ...
Source: Springer protocols feed by Microbiology - February 13, 2015 Category: Microbiology Source Type: news
Monitoring Translocation of Multisubunit RNA Polymerase Along the DNA with Fluorescent Base Analogues
We describe two template DNA strand designs where translocation of RNA polymerase from a pre-translocation to a post-translocation state results in disruption of stacking interactions of fluorophore with neighboring bases, with a concomitant large increase in fluorescence intensity. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - February 13, 2015 Category: Microbiology Source Type: news
Purification of Bacterial RNA Polymerase: Tools and Protocols
Bacterial RNA polymerase is the first point of gene expression and a validated target for antibiotics. Studied for several decades, the Escherichia coli transcriptional apparatus is by far the best characterized, with numerous RNA polymerase mutants and auxiliary factors isolated and analyzed in great detail. Since the E. coli enzyme was refractory to crystallization, structural studies have been focused on Thermus RNA polymerases, revealing atomic details of the catalytic center and RNA polymerase interactions with nucleic acids, antibiotics, and regulatory proteins. However, numerous differences between these enzymes, in...
Source: Springer protocols feed by Microbiology - February 13, 2015 Category: Microbiology Source Type: news
ChIP-Seq for Genome-Scale Analysis of Bacterial DNA-Binding Proteins
Protein–DNA interactions are central to many basic biological processes, including transcription regulation, DNA replication, and DNA repair. Chromatin Immunoprecipitation (ChIP) is used to determine the position and strength of protein–DNA interactions in vivo. Coupling ChIP with microarrays (ChIP-chip), and more recently with deep sequencing (ChIP-seq), has allowed genome-wide profiling of DNA binding events in vivo. In this chapter we outline the steps to generate ChIP-seq libraries from bacterial samples and briefly discuss basic analysis of the data. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - February 13, 2015 Category: Microbiology Source Type: news
Mapping the Escherichia coli Transcription Elongation Complex with Exonuclease III
RNA polymerase interactions with the nucleic acids control every step of the transcription cycle. These contacts mediate RNA polymerase recruitment to promoters, induce pausing during RNA chain synthesis, and control transcription termination. These interactions are dissected using footprinting assays, in which a bound protein protects nucleic acids from the digestion by nucleases or modification by chemical probes. Exonuclease III is frequently employed to study protein–DNA interactions owing to relatively simple procedures and low background. Exonuclease III has been used to determine RNA polymerase position in tra...
Source: Springer protocols feed by Microbiology - February 13, 2015 Category: Microbiology Source Type: news
Experimental Analysis of hFACT Action During Pol II Transcription In Vitro
We present the protocols describing how to prepare different forms of nucleosomes, including intact nucleosome, covalently conjugated nucleosome, nucleosome missing one of the two H2A/2B dimers (hexasome) and tetrasome (a nucleosome missing both H2A/2B dimers). These complexes allow analysis of various aspects of FACT’s function. These approaches and other methods described below can also be applied to the study of other chromatin remodelers and chromatin-targeted factors. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - February 13, 2015 Category: Microbiology Source Type: news
Transcription in Archaea: In Vitro Transcription Assays for mjRNAP
The fully recombinant Methanocaldococcus jannaschii RNA polymerase allows for a detailed dissection of the different stages of the transcription. In the previous chapter, we discussed how to purify the different components of the M. jannaschii transcription system, the RNA polymerase subunits, and general transcription factors and how to assemble a functional M. jannaschii enzyme. Standard in vitro transcription assays can be used to examine the different stages of transcription. In this chapter, we describe how some of these assays have been optimized for M. jannaschii RNA polymerase, which transcribes at much higher temp...
Source: Springer protocols feed by Microbiology - February 13, 2015 Category: Microbiology Source Type: news
Transcription in Archaea: Preparation of Methanocaldococcus jannaschii Transcription Machinery
Archaeal RNA polymerase and general transcription factors are more closely related to those of eukaryotes than of bacteria. As such the study of transcription of archaea is important both in terms of examination of the evolution of the transcriptional machinery and as a simplified tool for eukaryotic transcription. In particular, the hyperthermophilic Methanocaldococcus jannaschii provides us with a fully recombinant RNA polymerase system allowing for much more detailed in vitro examination of the roles of different components during the transcription cycle than otherwise possible. The individual subunits of M. jannaschii ...
Source: Springer protocols feed by Microbiology - February 13, 2015 Category: Microbiology Source Type: news
Purification of Active RNA Polymerase I from Yeast
Eukaryotic cells employ at least three nuclear, DNA-dependent RNA polymerase systems for the synthesis of cellular RNA. RNA polymerases I, II, and III primarily produce rRNA, mRNA, and tRNA, respectively. In a rapidly growing cell, most RNA synthesis is devoted to production of the translation machinery, with rRNA synthesis by RNA polymerase I representing more than half of total cellular transcription. The fundamental connection between ribosome biogenesis and cell growth is clear; furthermore, recent studies have identified transcription by RNA polymerase I as a key target for anticancer chemotherapy. Thus, efficient met...
Source: Springer protocols feed by Microbiology - February 13, 2015 Category: Microbiology Source Type: news
Manipulating Archaeal Systems to Permit Analyses of Transcription Elongation-Termination Decisions In Vitro
Transcription elongation by multisubunit RNA polymerases (RNAPs) is processive, but neither uniform nor continuous. Regulatory events during elongation include pausing, backtracking, arrest, and transcription termination, and it is critical to determine whether the absence of continued synthesis is transient or permanent. Here we describe mechanisms to generate large quantities of stable archaeal elongation complexes on a solid support to permit (1) single-round transcription, (2) walking of RNAP to any defined template position, and (3) discrimination of transcripts that are associated with RNAP from those that are releas...
Source: Springer protocols feed by Microbiology - February 13, 2015 Category: Microbiology Source Type: news
Using Solutes and Kinetics to Probe Large Conformational Changes in the Steps of Transcription Initiation
Small solutes are useful probes of large conformational changes in RNA polymerase–promoter interactions and other biopolymer processes. In general, a large effect of a solute on an equilibrium constant (or rate constant) indicates a large change in water-accessible biopolymer surface area in the corresponding step (or transition state), resulting from conformational changes, interface formation, or both. Here, we describe nitrocellulose filter binding assays from series used to determine the urea dependence of open complex formation and dissociation with Escherichia coli RNA polymerase and phage λPR promoter D...
Source: Springer protocols feed by Microbiology - February 13, 2015 Category: Microbiology Source Type: news
In Situ Footprinting of E. coli Transcription Elongation Complex with Chloroacetaldehyde
The structure and dynamics of Escherichia coli transcription elongation complex are now well documented. However, most of the studies have been conducted in vitro and frequently under artificial conditions that facilitate the biochemical characterization of the complex. Thus, little is known about relevance of these results for the regulatory aspects of transcription elongation inside the cell. Here, we describe the use of a single-strand-specific probe chloroacetaldehyde for in situ footprinting of E. coli elongation complex temporarily halted by a protein roadblock. The method provides valuable information on the dynamic...
Source: Springer protocols feed by Microbiology - February 13, 2015 Category: Microbiology Source Type: news
Preparation of cDNA Libraries for High-Throughput RNA Sequencing Analysis of RNA 5′ Ends
We provide a detailed protocol for preparing cDNA libraries suitable for high-throughput sequencing that are derived specifically from the 5′ ends of RNA (5′ specific RNA-seq). The protocol describes how cDNA libraries for 5′ specific RNA-seq can be tailored to analyze specific classes of RNAs based upon the phosphorylation status of the 5′ end. Thus, the analysis of cDNA libraries generated by these methods provides information regarding both the sequence and phosphorylation status of the 5′ ends of RNAs. 5′ specific RNA-seq can be used to analyze transcription initiation and posttransc...
Source: Springer protocols feed by Microbiology - February 13, 2015 Category: Microbiology Source Type: news
