Structural study of an RNA polymerase subunit unique for
gram-positive bacteria

a 2009

Structural study of an RNA polymerase subunit unique for gram-positive bacteria

MOTÁČKOVÁ, Veronika; Hana ŠANDEROVÁ; Jiří NOVÁČEK; Lukáš ŽÍDEK; Libor KRÁSNÝ et al.

Základní údaje

Originální název

Structural study of an RNA polymerase subunit unique for gram-positive bacteria

Název česky

Strukturní studie podjednotky RNA polymerasy specifické pro gram-positivní bakterie

Název anglicky

Structural study of an RNA polymerase subunit unique for gram-positive bacteria

Autoři

MOTÁČKOVÁ, Veronika; Hana ŠANDEROVÁ; Jiří NOVÁČEK; Lukáš ŽÍDEK; Libor KRÁSNÝ a Vladimír SKLENÁŘ

Vydání

Central European NMR Meeting Valtice, 2009

Další údaje

Typ výsledku

Konferenční abstrakt

Utajení

není předmětem státního či obchodního tajemství

Označené pro přenos do RIV

Organizační jednotka

Přírodovědecká fakulta

ISBN

978-80-86441-40-5

Klíčová slova anglicky

nmr, rna polymerase, assignment, structure, validation

Příznaky

Mezinárodní význam

Změněno: 30. 4. 2009 12:46, Mgr. Veronika Papoušková, Ph.D.

Anotace

V originále

RNA polymerases (RNAPs) from gram-positive bacteria of Bacillus subtilis differ from well-studied RNA polymerases from gram-negative bacteria in the presence of two additional subunits interacting with the core enzyme, delta and omega1. Their role in the transcription machinery is not well understood. Recent reports indicating the importance of the delta subunit for the virulence of Staphylococcus aureus and Streptococcus agalactiae make the delta subunit interesting from the medical point of view. Although the influence of the delta subunit on the activity of RNA polymerase was indicated many years ago, its structure and function remained unknown. The C-terminal domain of the protein is mostly unstructured and highly negatively charged. That might mimic nucleic acids and compete with DNA/RNA for binding on RNAP. The well-structured N-terminal domain is proposed to bind to the RNAP core and probably orients the C-terminal domain on the surface of RNAP. Preliminary results showed that signals of atoms from a separate N-terminal domain overlap with corresponding peaks in spectra of the whole delta subunit. Therefore, we decided to focus on the well-ordered N-terminal domain to determine its structure using nuclear magnetic resonance (NMR). A standard set of triple resonance NMR experiments was measured and all resonances of the protein backbone were assigned. Resonance frequencies of the side-chains were assigned using 3D TOCSY- and NOESY-type spectra. Three-bond coupling constant 3J(HNHA) were obtained from 3D HNHA experiments. Chemical shifts of backbone nuclei, medium range NOEs, and the three-bond coupling constants were analyzed and secondary structure was predicted. Internuclear distance restraints were extracted from NOESY spectra. All backbone and side-chain resonance frequencies were assigned using the standard methodology. Secondary structure was predicted based on medium-range NOEs, J(HNHA), and chemical shifts, analyzed by programs CSI and TALOS. The NOE cross-peaks were assigned using program ARIA 2.1 The unambiguously assigned distance, torsion angle, and hydrogen bond restraints were used in the final refinement in program CNS with input scripts from the authors of the RECOORD database. Programs PROCHECK and WHATIF were used to check the quality of the calculated structures.

Návaznosti

GA204/09/0583, projekt VaV

Název: Delta podjednotka RNA polymerázy z Gram pozitivních bakterií (Akronym: DELTA)

Investor: Grantová agentura ČR, Delta podjednotka RNA polymerázy z Gram pozitivních bakterií

LC06030, projekt VaV

Název: Biomolekulární centrum

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Biomolekulární centrum

MSM0021622413, záměr

Název: Proteiny v metabolismu a při interakci organismů s prostředím

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Proteiny v metabolismu a při interakci organismů s prostředím

Citovat

MOTÁČKOVÁ, Veronika; Hana ŠANDEROVÁ; Jiří NOVÁČEK; Lukáš ŽÍDEK; Libor KRÁSNÝ a Vladimír SKLENÁŘ. Structural study of an RNA polymerase subunit unique for gram-positive bacteria. In Central European NMR Meeting Valtice. 2009. ISBN 978-80-86441-40-5.

@proceedings{830083,
   author = {Motáčková, Veronika and Šanderová, Hana and Nováček, Jiří and Žídek, Lukáš and Krásný, Libor and Sklenář, Vladimír},
   booktitle = {Central European NMR Meeting Valtice},
   keywords = {nmr, rna polymerase, assignment, structure, validation},
   isbn = {978-80-86441-40-5},
   title = {Structural study of an RNA polymerase subunit unique for gram-positive bacteria},
   year = {2009}
}

TY  - CONF
ID  - 830083
AU  - Motáčková, Veronika - Šanderová, Hana - Nováček, Jiří - Žídek, Lukáš - Krásný, Libor - Sklenář, Vladimír
PY  - 2009
TI  - Structural study of an RNA polymerase subunit unique for gram-positive bacteria
SN  - 9788086441405
KW  - nmr, rna polymerase, assignment, structure, validation
N2  - RNA polymerases (RNAPs) from gram-positive bacteria of Bacillus subtilis differ from well-studied RNA polymerases from gram-negative bacteria in the presence of two additional subunits interacting with the core enzyme, delta and omega1. Their role in the transcription machinery is not well understood. Recent reports indicating the importance of the delta subunit for the virulence of Staphylococcus aureus and Streptococcus agalactiae make the delta subunit interesting from the medical point of view. Although the influence of the delta subunit on the activity of RNA polymerase was indicated many years ago, its structure and function remained unknown. The C-terminal domain of the protein is mostly unstructured and highly negatively charged. That might mimic nucleic acids and compete with DNA/RNA for binding on RNAP. The well-structured N-terminal domain is proposed to bind to the RNAP core and probably orients the C-terminal domain on the surface of RNAP. Preliminary results showed that signals of atoms from a separate N-terminal domain overlap with corresponding peaks in spectra of the whole delta subunit. Therefore, we decided to focus on the well-ordered N-terminal domain to determine its structure using nuclear magnetic resonance (NMR). A standard set of triple resonance NMR experiments was measured and all resonances of the protein backbone were assigned. Resonance frequencies of the side-chains were assigned using 3D TOCSY- and NOESY-type spectra. Three-bond coupling constant 3J(HNHA) were obtained from 3D HNHA experiments. Chemical shifts of backbone nuclei, medium range NOEs, and the three-bond coupling constants were analyzed and secondary structure was predicted. Internuclear distance restraints were extracted from NOESY spectra. All backbone and side-chain resonance frequencies were assigned using the standard methodology. Secondary structure was predicted based on medium-range NOEs, J(HNHA), and chemical shifts, analyzed by programs CSI and TALOS. The NOE cross-peaks were assigned using program ARIA 2.1 The unambiguously assigned distance, torsion angle, and hydrogen bond restraints were used in the final refinement in program CNS with input scripts from the authors of the RECOORD database. Programs PROCHECK and WHATIF were used to check the quality of the calculated structures.
ER  -

MOTÁČKOVÁ, Veronika; Hana ŠANDEROVÁ; Jiří NOVÁČEK; Lukáš ŽÍDEK; Libor KRÁSNÝ a Vladimír SKLENÁŘ. Structural study of an RNA polymerase subunit unique for gram-positive bacteria. In \textit{Central European NMR Meeting Valtice}. 2009. ISBN~978-80-86441-40-5.

Přehled o publikaci