scholarly journals The stress sigma factor of RNA polymerase RpoS/σS is a solvent-exposed open molecule in solution

2018 ◽  
Vol 475 (1) ◽  
pp. 341-354 ◽  
Author(s):  
Paola Cavaliere ◽  
Sébastien Brier ◽  
Petr Filipenko ◽  
Christina Sizun ◽  
Bertrand Raynal ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Analytical Ultracentrifugation ◽  
Current Model ◽  
Three Dimensional ◽  
Global Gene Expression ◽  
Dimensional Structure ◽  
Bacterial Survival ◽  
Promoter Recognition ◽  
Σ Factor ◽  
Compact Conformation

In bacteria, one primary and multiple alternative sigma (σ) factors associate with the RNA polymerase core enzyme (E) to form holoenzymes (Eσ) with different promoter recognition specificities. The alternative σ factor RpoS/σS is produced in stationary phase and under stress conditions and reprograms global gene expression to promote bacterial survival. To date, the three-dimensional structure of a full-length free σ factor remains elusive. The current model suggests that extensive interdomain contacts in a free σ factor result in a compact conformation that masks the DNA-binding determinants of σ, explaining why a free σ factor does not bind double-stranded promoter DNA efficiently. Here, we explored the solution conformation of σS using amide hydrogen/deuterium exchange coupled with mass spectrometry, NMR, analytical ultracentrifugation and molecular dynamics. Our data strongly argue against a compact conformation of free σS. Instead, we show that σS adopts an open conformation in solution in which the folded σ2 and σ4 domains are interspersed by domains with a high degree of disorder. These findings suggest that E binding induces major changes in both the folding and domain arrangement of σS and provide insights into the possible mechanisms of regulation of σS activity by its chaperone Crl.

Assembly states of the nucleosome assembly protein 1 (NAP-1) revealed by sedimentation velocity and non-denaturing MS

2011 ◽  
Vol 436 (1) ◽  
pp. 101-112 ◽  
Author(s):  
Masanori Noda ◽  
Susumu Uchiyama ◽  
Adam R. McKay ◽  
Akihiro Morimoto ◽  
Shigeki Misawa ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Electrostatic Interactions ◽  
Analytical Ultracentrifugation ◽  
Three Dimensional ◽  
Sedimentation Velocity ◽  
Dimensional Structure ◽  
Histone H2a ◽  
Nucleosome Assembly ◽  
Nucleosome Assembly Protein ◽  
Nucleosome Assembly Protein 1

Proteins often exist as ensembles of interconverting states in solution which are often difficult to quantify. In the present manuscript we show that the combination of MS under nondenaturing conditions and AUC-SV (analytical ultracentrifugation sedimentation velocity) unambiguously clarifies a distribution of states and hydrodynamic shapes of assembled oligomers for the NAP-1 (nucleosome assembly protein 1). MS established the number of associated units, which was utilized as input for the numerical analysis of AUC-SV profiles. The AUC-SV analysis revealed that less than 1% of NAP-1 monomer exists at the micromolar concentration range and that the basic assembly unit consists of dimers of yeast or human NAP-1. These dimers interact non-covalently to form even-numbered higher-assembly states, such as tetramers, hexamers, octamers and decamers. MS and AUC-SV consistently showed that the formation of the higher oligomers was suppressed with increasing ionic strength, implicating electrostatic interactions in the formation of higher oligomers. The hydrodynamic shapes of the NAP-1 tetramer estimated from AUC-SV agreed with the previously proposed assembly models built using the known three-dimensional structure of yeast NAP-1. Those of the hexamer and octamer could be represented by new models shown in the present study. Additionally, MS was used to measure the stoichiometry of the interaction between the human NAP-1 dimer and the histone H2A–H2B dimer or H3–H4 tetramer. The present study illustrates a rigorous procedure for the analysis of protein assembly and protein–protein interactions in solution.

scholarly journals Bacterial RNA polymerase can retain σ70 throughout transcription

2016 ◽  
Vol 113 (3) ◽  
pp. 602-607 ◽  
Author(s):  
Timothy T. Harden ◽  
Christopher D. Wells ◽  
Larry J. Friedman ◽  
Robert Landick ◽  
Ann Hochschild ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Single Molecule ◽  
Transcription Initiation ◽  
Messenger Rna ◽  
Initiation Factors ◽  
Bacterial Rna ◽  
Direct Measurements ◽  
Promoter Recognition ◽  
Σ Factor ◽  
Transcript Elongation

Production of a messenger RNA proceeds through sequential stages of transcription initiation and transcript elongation and termination. During each of these stages, RNA polymerase (RNAP) function is regulated by RNAP-associated protein factors. In bacteria, RNAP-associated σ factors are strictly required for promoter recognition and have historically been regarded as dedicated initiation factors. However, the primary σ factor in Escherichia coli, σ70, can remain associated with RNAP during the transition from initiation to elongation, influencing events that occur after initiation. Quantitative studies on the extent of σ70 retention have been limited to complexes halted during early elongation. Here, we used multiwavelength single-molecule fluorescence-colocalization microscopy to observe the σ70–RNAP complex during initiation from the λ PR′ promoter and throughout the elongation of a long (>2,000-nt) transcript. Our results provide direct measurements of the fraction of actively transcribing complexes with bound σ70 and the kinetics of σ70 release from actively transcribing complexes. σ70 release from mature elongation complexes was slow (0.0038 s−1); a substantial subpopulation of elongation complexes retained σ70 throughout transcript elongation, and this fraction depended on the sequence of the initially transcribed region. We also show that elongation complexes containing σ70 manifest enhanced recognition of a promoter-like pause element positioned hundreds of nucleotides downstream of the promoter. Together, the results provide a quantitative framework for understanding the postinitiation roles of σ70 during transcription.

scholarly journals The Structure of Bypass of Forespore C, an Intercompartmental Signaling Factor during Sporulation in Bacillus

2005 ◽  
Vol 280 (43) ◽  
pp. 36214-36220 ◽  
Author(s):  
Hayley M. Patterson ◽  
James A. Brannigan ◽  
Simon M. Cutting ◽  
Keith S. Wilson ◽  
Anthony J. Wilkinson ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Mother Cell ◽  
Asymmetric Cell Division ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Coat Proteins ◽  
Terminal Domain ◽  
Spore Coat Proteins ◽  
Α Helix ◽  
Β Sheet

Sporulation in Bacillus subtilis begins with an asymmetric cell division giving rise to smaller forespore and larger mother cell compartments. Different programs of gene expression are subsequently directed by compartment-specific RNA polymerase σ-factors. In the final stages, spore coat proteins are synthesized in the mother cell under the control of RNA polymerase containing σK, (EσK). σK is synthesized as an inactive zymogen, pro-σK, which is activated by proteolytic cleavage. Processing of pro-σK is performed by SpoIVFB, a metalloprotease that resides in a complex with SpoIVFA and bypass of forespore (Bof)A in the outer forespore membrane. Ensuring coordination of events taking place in the two compartments, pro-σK processing in the mother cell is delayed until appropriate signals are received from the forespore. Cell-cell signaling is mediated by SpoIVB and BofC, which are expressed in the forespore and secreted to the intercompartmental space where they regulate pro-σK processing by mechanisms that are not yet fully understood. Here we present the three-dimensional structure of BofC determined by solution state NMR. BofC is a monomer made up of two domains. The N-terminal domain, containing a four-stranded β-sheet onto one face of which an α-helix is packed, closely resembles the third immunoglobulin-binding domain of protein G from Streptococcus. The C-terminal domain contains a three-stranded β-sheet and three α-helices in a novel domain topology. The sequence connecting the domains contains a conserved DISP motif to which mutations that affect BofC activity map. Possible roles for BofC in the σK checkpoint are discussed in the light of sequence and structure comparisons.

scholarly journals Bacteriophage T4 Genome

2003 ◽  
Vol 67 (1) ◽  
pp. 86-156 ◽  
Author(s):  
Eric S. Miller ◽  
Elizabeth Kutter ◽  
Gisela Mosig ◽  
Fumio Arisaka ◽  
Takashi Kunisawa ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Functional Genomics ◽  
Genomic Sequence ◽  
Bacteriophage T4 ◽  
Three Dimensional ◽  
Tail Fiber ◽  
Structural Level ◽  
Genomics And Proteomics ◽  
Transcriptional Pattern ◽  
Promoter Recognition

SUMMARY Phage T4 has provided countless contributions to the paradigms of genetics and biochemistry. Its complete genome sequence of 168,903 bp encodes about 300 gene products. T4 biology and its genomic sequence provide the best-understood model for modern functional genomics and proteomics. Variations on gene expression, including overlapping genes, internal translation initiation, spliced genes, translational bypassing, and RNA processing, alert us to the caveats of purely computational methods. The T4 transcriptional pattern reflects its dependence on the host RNA polymerase and the use of phage-encoded proteins that sequentially modify RNA polymerase; transcriptional activator proteins, a phage sigma factor, anti-sigma, and sigma decoy proteins also act to specify early, middle, and late promoter recognition. Posttranscriptional controls by T4 provide excellent systems for the study of RNA-dependent processes, particularly at the structural level. The redundancy of DNA replication and recombination systems of T4 reveals how phage and other genomes are stably replicated and repaired in different environments, providing insight into genome evolution and adaptations to new hosts and growth environments. Moreover, genomic sequence analysis has provided new insights into tail fiber variation, lysis, gene duplications, and membrane localization of proteins, while high-resolution structural determination of the “cell-puncturing device,” combined with the three-dimensional image reconstruction of the baseplate, has revealed the mechanism of penetration during infection. Despite these advances, nearly 130 potential T4 genes remain uncharacterized. Current phage-sequencing initiatives are now revealing the similarities and differences among members of the T4 family, including those that infect bacteria other than Escherichia coli. T4 functional genomics will aid in the interpretation of these newly sequenced T4-related genomes and in broadening our understanding of the complex evolution and ecology of phages—the most abundant and among the most ancient biological entities on Earth.

scholarly journals Novel insertion and deletion mutants of RpoB that render Mycobacterium smegmatis RNA polymerase resistant to rifampicin-mediated inhibition of transcription

Microbiology ◽  
2010 ◽  
Vol 156 (5) ◽  
pp. 1565-1573 ◽  
Author(s):  
Vidyasagar Malshetty ◽  
Krishna Kurthkoti ◽  
Arnab China ◽  
Bratati Mallick ◽  
Subburaj Yamunadevi ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Mycobacterium Smegmatis ◽  
Three Dimensional ◽  
Rna Polymerases ◽  
Dimensional Structure ◽  
Deletion Mutants ◽  
Insertion And Deletion ◽  
Calf Thymus ◽  
Specific Activities ◽  
Spatial Positioning

The startling increase in the occurrence of rifampicin (Rif) resistance in the clinical isolates of Mycobacterium tuberculosis worldwide is posing a serious concern to tuberculosis management. The majority of Rif resistance in bacteria arises from mutations in the RpoB subunit of the RNA polymerase. We isolated M. smegmatis strains harbouring either an insertion (6 aa) or a deletion (10 aa) in their RpoB proteins. Although these strains showed a compromised fitness for growth in 7H9 Middlebrook medium, their resistance to Rif was remarkably high. The attenuated growth of the strains correlated with decreased specific activities of the RNA polymerases from the mutants. While the RNA polymerases from the parent or a mutant strain (harbouring a frequently occurring mutation, H442Y, in RpoB) were susceptible to Rif-mediated inhibition of transcription from calf thymus DNA, those from the insertion and deletion mutants were essentially refractory to such inhibition. Three-dimensional structure modelling revealed that the RpoB amino acids that interact with Rif are either deleted or unable to interact with Rif due to their unsuitable spatial positioning in these mutants. We discuss possible uses of the RpoB mutants in studying transcriptional regulation in mycobacteria and as potential targets for drug design.

Crystal structure and kinetic study of dihydrodipicolinate synthase from Mycobacterium tuberculosis

2008 ◽  
Vol 411 (2) ◽  
pp. 351-360 ◽  
Author(s):  
Georgia Kefala ◽  
Genevieve L. Evans ◽  
Michael D. W. Griffin ◽  
Sean R. A. Devenish ◽  
F. Grant Pearce ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Active Site ◽  
Analytical Ultracentrifugation ◽  
Feedback Inhibition ◽  
Three Dimensional ◽  
Lysine Biosynthesis ◽  
Dimensional Structure ◽  
Dihydrodipicolinate Synthase ◽  
Three Dimensional Structure ◽  
And Function

The three-dimensional structure of the enzyme dihydrodipicolinate synthase (KEGG entry Rv2753c, EC 4.2.1.52) from Mycobacterium tuberculosis (Mtb-DHDPS) was determined and refined at 2.28 Å (1 Å=0.1 nm) resolution. The asymmetric unit of the crystal contains two tetramers, each of which we propose to be the functional enzyme unit. This is supported by analytical ultracentrifugation studies, which show the enzyme to be tetrameric in solution. The structure of each subunit consists of an N-terminal (β/α)8-barrel followed by a C-terminal α-helical domain. The active site comprises residues from two adjacent subunits, across an interface, and is located at the C-terminal side of the (β/α)8-barrel domain. A comparison with the other known DHDPS structures shows that the overall architecture of the active site is largely conserved, albeit the proton relay motif comprising Tyr143, Thr54 and Tyr117 appears to be disrupted. The kinetic parameters of the enzyme are reported: KMASA=0.43±0.02 mM, KMpyruvate=0.17±0.01 mM and Vmax=4.42±0.08 μmol·s−1·mg−1. Interestingly, the Vmax of Mtb-DHDPS is 6-fold higher than the corresponding value for Escherichia coli DHDPS, and the enzyme is insensitive to feedback inhibition by (S)-lysine. This can be explained by the three-dimensional structure, which shows that the (S)-lysine-binding site is not conserved in Mtb-DHDPS, when compared with DHDPS enzymes that are known to be inhibited by (S)-lysine. A selection of metabolites from the aspartate family of amino acids do not inhibit this enzyme. A comprehensive understanding of the structure and function of this important enzyme from the (S)-lysine biosynthesis pathway may provide the key for the design of new antibiotics to combat tuberculosis.

scholarly journals Structural and Biophysical Studies on Two Promoter Recognition Domains of the Extra-cytoplasmic Function σ Factor σC from Mycobacterium tuberculosis

2006 ◽  
Vol 282 (7) ◽  
pp. 4711-4718 ◽  
Author(s):  
Krishan Gopal Thakur ◽  
Anagha Madhusudan Joshi ◽  
B. Gopal
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rna Polymerase ◽  
Mutational Analysis ◽  
Sequence Similarity ◽  
Regulation Of Transcription ◽  
Transcriptional Regulatory ◽  
Promoter Recognition ◽  
Interaction Interaction ◽  
Σ Factor ◽  
Pribnow Box

σ factors are transcriptional regulatory proteins that bind to the RNA polymerase and dictate gene expression. The extracytoplasmic function (ECF) σ factors govern the environment dependent regulation of transcription. ECF σ factors have two domains σ2 and σ4 that recognize the -10 and -35 promoter elements. However, unlike the primary σ factor σA, the ECF σ factors lack σ3, a region that helps in the recognition of the extended -10 element and σ1.1, a domain involved in the autoinhibition of σA in the absence of core RNA polymerase. Mycobacterium tuberculosis σC is an ECF σ factor that is essential for the pathogenesis and virulence of M. tuberculosis in the mouse and guinea pig models of infection. However, unlike other ECF σ factors, σC does not appear to have a regulatory anti-σ factor located in the same operon. We also note that M. tuberculosis σC differs from the canonical ECF σ factors as it has an N-terminal domain comprising of 126 amino acids that precedes the σC2 and σC4 domains. In an effort to understand the regulatory mechanism of this protein, the crystal structures of the σC2 and σC4 domains of σC were determined. These promoter recognition domains are structurally similar to the corresponding domains of σA despite the low sequence similarity. Fluorescence experiments using the intrinsic tryptophan residues of σC2 as well as surface plasmon resonance measurements reveal that the σC2 and σC4 domains interact with each other. Mutational analysis suggests that the Pribnow box-binding region of σC2 is involved in this interdomain interaction. Interaction between the promoter recognition domains in M. tuberculosis σC are thus likely to regulate the activity of this protein even in the absence of an anti-σ factor.

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