Antibody inhibition of the RNA polymerase of a rotavirus: A cryoelectron microscopy and X-ray crystallography study

1998 ◽  
Vol 90 (3) ◽  
pp. 281-281
Author(s):  
Eric Thouvenin ◽  
Elizabeth Hewat ◽  
Guy Schoehn ◽  
Félix Rey ◽  
Isabelle Petitpas ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Cryoelectron Microscopy ◽  
X Ray ◽  
Rotavirus A ◽  
X Ray Crystallography ◽  
Antibody Inhibition

scholarly journals Structure of human RNA Polymerase III

2020 ◽  
Author(s):  
Ewan Phillip Ramsay ◽  
Guillermo Abascal-Palacios ◽  
Julia L. Daiß ◽  
Helen King ◽  
Jerome Gouge ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Cellular Growth ◽  
Loss Of Function ◽  
Critical Determinant ◽  
Saxs Data ◽  
X Ray ◽  
X Ray Crystallography ◽  
The Stability ◽  
Pol Iii

ABSTRACTIn eukaryotes, RNA Polymerase (Pol) III is the enzyme specialised for the transcription of the entire pool of tRNAs and several other short, essential, untranslated RNAs. Pol III is a critical determinant of cellular growth and lifespan across the eukaryotic kingdom. Upregulation of Pol III transcription is often observed in cancer cells and causative Pol III mutations have been described in patients affected by severe neurodevelopmental disorders and hypersensitivity to viral infection.Harnessing CRISPR-Cas9 genome editing in HeLa cells, we isolated endogenous human Pol III and obtained a cryo-EM reconstruction at 4.0 Å. The structure of human Pol III allowed us to map the reported genetic mutations and rationalise them. Mutations causing neurodevelopmental defects cluster in hotspots that affect the stability and/or biogenesis of Pol III, thereby resulting in loss-of-function of the enzyme. Mutations affecting viral sensing are located in the periphery of the enzyme in proximity to DNA binding regions, suggesting an impairment of Pol III cytosolic viral DNA-sensing activity.Furthermore, integrating x-ray crystallography and SAXS data, we describe the structure of the RPC5 C-terminal extension, which is absent in lower eukaryotes and not visible in our EM map. Surprisingly, experiments in living cells highlight a role for the RPC5 C-terminal extension in the correct assembly and stability of the human Pol III enzyme, thus suggesting an added layer of regulation during the biogenesis of Pol III in higher eukaryotes.

scholarly journals The Structure of Cucumber Mosaic Virus: Cryoelectron Microscopy, X-Ray Crystallography, and Sequence Analysis

Virology ◽  
1997 ◽  
Vol 232 (1) ◽  
pp. 91-97 ◽  
Author(s):  
William R. Wikoff ◽  
Chao Jo Tsai ◽  
Guoji Wang ◽  
Timothy S. Baker ◽  
John E. Johnson
Keyword(s):  
Sequence Analysis ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Cryoelectron Microscopy ◽  
X Ray ◽  
X Ray Crystallography

Direct Imaging of Interactions between an Icosahedral Virus and Conjugate Fab Fragments by Cryoelectron Microscopy and X-Ray Crystallography

Virology ◽  
1994 ◽  
Vol 204 (2) ◽  
pp. 777-788 ◽  
Author(s):  
Claudine Porta ◽  
Guoji Wang ◽  
Holland Cheng ◽  
Zhongguo Chen ◽  
Timothy S. Baker ◽  
...  
Keyword(s):  
Cryoelectron Microscopy ◽  
Direct Imaging ◽  
X Ray ◽  
X Ray Crystallography ◽  
Fab Fragments ◽  
Icosahedral Virus

scholarly journals Structure of human RNA polymerase III

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ewan Phillip Ramsay ◽  
Guillermo Abascal-Palacios ◽  
Julia L. Daiß ◽  
Helen King ◽  
Jerome Gouge ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Neurodevelopmental Disorders ◽  
Rna Polymerase Iii ◽  
Living Cells ◽  
Cellular Growth ◽  
Genetic Mutations ◽  
Viral Dna ◽  
X Ray ◽  
X Ray Crystallography ◽  
Pol Iii

AbstractIn eukaryotes, RNA Polymerase (Pol) III is specialized for the transcription of tRNAs and other short, untranslated RNAs. Pol III is a determinant of cellular growth and lifespan across eukaryotes. Upregulation of Pol III transcription is observed in cancer and causative Pol III mutations have been described in neurodevelopmental disorders and hypersensitivity to viral infection. Here, we report a cryo-EM reconstruction at 4.0 Å of human Pol III, allowing mapping and rationalization of reported genetic mutations. Mutations causing neurodevelopmental defects cluster in hotspots affecting Pol III stability and/or biogenesis, whereas mutations affecting viral sensing are located in proximity to DNA binding regions, suggesting an impairment of Pol III cytosolic viral DNA-sensing. Integrating x-ray crystallography and SAXS, we also describe the structure of the higher eukaryote specific RPC5 C-terminal extension. Surprisingly, experiments in living cells highlight a role for this module in the assembly and stability of human Pol III.

scholarly journals Identification of a Fab interaction footprint site on an icosahedral virus by cryoelectron microscopy and X-ray crystallography

Nature ◽  
1992 ◽  
Vol 355 (6357) ◽  
pp. 275-278 ◽  
Author(s):  
Guoji Wang ◽  
Claudine Porta ◽  
Zhongguo Chen ◽  
Timothy S. Baker ◽  
John E. Johnson
Keyword(s):  
Cryoelectron Microscopy ◽  
X Ray ◽  
X Ray Crystallography ◽  
Icosahedral Virus

scholarly journals Antibody-Mediated Neutralization of Human Rhinovirus 14 Explored by Means of Cryoelectron Microscopy and X-Ray Crystallography of Virus-Fab Complexes

1998 ◽  
Vol 72 (6) ◽  
pp. 4610-4622 ◽  
Author(s):  
Zhiwei Che ◽  
Norman H. Olson ◽  
Donna Leippe ◽  
Wai-ming Lee ◽  
Anne G. Mosser ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Significant Proportion ◽  
Human Rhinovirus ◽  
Intercellular Adhesion Molecule ◽  
Cryoelectron Microscopy ◽  
Intercellular Adhesion Molecule 1 ◽  
Sequence Comparisons ◽  
X Ray ◽  
X Ray Crystallography ◽  
South Wall

ABSTRACT The structures of three different human rhinovirus 14 (HRV14)-Fab complexes have been explored with X-ray crystallography and cryoelectron microscopy procedures. All three antibodies bind to the NIm-IA site of HRV14, which is the β-B–β-C loop of the viral capsid protein VP1. Two antibodies, Fab17-IA (Fab17) and Fab12-IA (Fab12), bind bivalently to the virion surface and strongly neutralize viral infectivity whereas Fab1-IA (Fab1) strongly aggregates and weakly neutralizes virions. The structures of the two classes of virion-Fab complexes clearly differ and correlate with observed binding neutralization differences. Fab17 and Fab12 bind in essentially identical, tangential orientations to the viral surface, which favors bidentate binding over icosahedral twofold axes. Fab1 binds in a more radial orientation that makes bidentate binding unlikely. Although the binding orientations of these two antibody groups differ, nearly identical charge interactions occur at all paratope-epitope interfaces. Nucleotide sequence comparisons suggest that Fab17 and Fab12 are from the same progenitor cell and that some of the differing residues contact the south wall of the receptor binding canyon that encircles each of the icosahedral fivefold vertices. All of the antibodies contact a significant proportion of the canyon region and directly overlap much of the receptor (intercellular adhesion molecule 1 [ICAM-1]) binding site. Fab1, however, does not contact the same residues on the upper south wall (the side facing away from fivefold axes) at the receptor binding region as do Fab12 and Fab17. All three antibodies cause some stabilization of HRV14 against pH-induced inactivation; thus, stabilization may be mediated by invariant contacts with the canyon.

scholarly journals The DNA binding domain of the Vibrio vulnificus SmcR transcription factor is flexible and recognizes diverse DNA sequences

2020 ◽  
Author(s):  
Jane D. Newman ◽  
Meghan M. Russell ◽  
Giovanni Gonzalez-Gutierrez ◽  
Julia C. van Kessel
Keyword(s):  
Dna Binding ◽  
Quorum Sensing ◽  
Rna Polymerase ◽  
Binding Site ◽  
Dna Sequences ◽  
Vibrio Vulnificus ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
X Ray ◽  
X Ray Crystallography

AbstractThe quorum-sensing regulon in vibrios is controlled by the LuxR/HapR family of transcriptional regulators. In Vibrio vulnificus, this regulator is called SmcR, and it controls expression of numerous virulence behaviors, including biofilm formation and elastase production. The consensus binding site of Vibrio LuxR/HapR/SmcR proteins is palindromic, as is common for regulators that bind as dimers with helix-turn-helix N-terminal DNA binding domains. However, the LuxR/HapR/SmcR consensus site is highly degenerate and asymmetric with variations in sequence at each promoter. To determine the mechanism of DNA site recognition, we generated separation-of-function mutants of SmcR that either repress or activate transcription but not both. The SmcR N55I protein is defective at transcription activation due to loss of binding to most DNA binding sites in activated promoters but retains interaction with RNA polymerase (RNAP) alpha. SmcR S76A, L139R, and N142D are defective for interaction with RNAP alpha but retain functional DNA binding activity. Using X-ray crystallography, we show that the wild-type SmcR dimer and the three RNAP-interaction mutants exhibit two conformations of the helix-turn-helix DNA binding domain. Conversely, the SmcR N55I X-ray crystal structure is limited to only one conformation and is restricted in recognition of single base-pair variations in DNA binding site sequences. These data support a model in which two mechanisms drive SmcR transcriptional activation: interaction with RNA polymerase and a multi-conformational DNA binding domain that permits recognition of variable DNA sites. Thus, the LuxR/HapR/SmcR proteins balance specificity for quorum-sensing targets and diversity to accommodate binding at hundreds of sites genome-wide.SignificanceThe cell-cell communication system called quorum sensing controls expression of genes required for virulence in Vibrio bacteria species, including the potent human pathogen Vibrio vulnificus. The master transcriptional regulator of quorum-sensing genes in vibrios belongs to the LuxR/HapR/SmcR family. These regulators directly activate and repress transcription of >100 genes via binding to degenerate sites in promoter regions. We used X-ray crystallography to determine the structure of mutant SmcR proteins. Our experiments reveal that SmcR recognizes diverse sequences via a DNA binding domain that samples multiple conformations to accommodate variations in palindromic DNA sequences. Significantly, the DNA binding domain of SmcR is completely conserved in LuxR/HapR/SmcR family proteins, suggesting that this mechanism is representative of quorum-sensing regulation in other vibrios.

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