Watching the Bacteriophage N4 RNA Polymerase Transcription by Time-dependent Soak-trigger-freeze X-ray Crystallography

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.

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Thioether linked meso functionalized BODIPY DYEmer

Journal of Porphyrins and Phthalocyanines ◽

10.1142/s1088424621500176 ◽

2021 ◽

pp. A-H

Author(s):

Bhausaheb Dhokale ◽

Indresh Singh Yadav ◽

Shaikh M. Mobin ◽

Rajneesh Misra

Keyword(s):

Dft Calculations ◽

Single Crystal ◽

Room Temperature ◽

Structural Parameters ◽

Time Dependent ◽

Lumo Energy ◽

X Ray ◽

X Ray Crystallography ◽

Td Dft ◽

Meso Position

Thioether linked meso BODIPY DYEmer 3 was synthesized by two different routes. The reaction of dipyrrothioketone 1 and 8-chloro BODIPY 2 in the presence of triethylamine followed by complexation with [Formula: see text] resulted in thioether linked meso functionalized BODIPY DYEmer 3. Using another route, the reaction of 8-chloro BODIPY 2 with sodium hydrosulphide (NaSH) at room temperature resulted in the thioether linked meso BODIPY DYEmer 3. The DYEmer 3 was characterized by 1H, [Formula: see text]C, [Formula: see text]B, [Formula: see text]F NMR, HRMS, and single crystal X-ray crystallography. The properties of DYEmer 3 was compared with the previously reported thioether linked [Formula: see text] and [Formula: see text] BODIPY DYEmers 4 and 5. The structural parameters indicating the intramolecular arrangements of two BODIPY units of DYEmer were compared and corelated with the observed properties. The time-dependent DFT (TD-DFT) calculations suggested that the thioether group at meso position of BODIPY 3 stabilizes the LUMO energy than 8-chloro BODIPY 2. Compared to 8-chloro BODIPY 2 the HOMO-1 of DYEmer 3 is destabilized whereas the LUMO+1 is stabilized.

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X-ray crystal structure of the polymerase domain of the bacteriophage N4 virion RNA polymerase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0712325105 ◽

2008 ◽

Vol 105 (13) ◽

pp. 5046-5051 ◽

Cited By ~ 15

Author(s):

Katsuhiko S. Murakami ◽

Elena K. Davydova ◽

Lucia B. Rothman-Denes

Keyword(s):

Crystal Structure ◽

Rna Polymerase ◽

X Ray ◽

Polymerase Domain ◽

Bacteriophage N4 ◽

N4 Virion

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Structure of human RNA polymerase III

Nature Communications ◽

10.1038/s41467-020-20262-5 ◽

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.

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

Biology of the Cell ◽

10.1016/s0248-4900(98)80050-6 ◽

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

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

10.1101/2020.10.30.362368 ◽

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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Difference Fourier Analysis of Glucose Embedded and Frozen Hydrated Purple Membrane

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100053164 ◽

1982 ◽

Vol 40 ◽

pp. 74-75

Author(s):

Jules S. Jaffe ◽

Robert M. Glaeser

Keyword(s):

Purple Membrane ◽

Data Set ◽

High Resolution Data ◽

X Ray ◽

X Ray Crystallography ◽

Fourier Techniques ◽

Versus Protein ◽

The Difference ◽

Difference Fourier ◽

Ideal Method

Although difference Fourier techniques are standard in X-ray crystallography it has only been very recently that electron crystallographers have been able to take advantage of this method. We have combined a high resolution data set for frozen glucose embedded Purple Membrane (PM) with a data set collected from PM prepared in the frozen hydrated state in order to visualize any differences in structure due to the different methods of preparation. The increased contrast between protein-ice versus protein-glucose may prove to be an advantage of the frozen hydrated technique for visualizing those parts of bacteriorhodopsin that are embedded in glucose. In addition, surface groups of the protein may be disordered in glucose and ordered in the frozen state. The sensitivity of the difference Fourier technique to small changes in structure provides an ideal method for testing this hypothesis.

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