Study of RNA polymerase delta subunit unique for gram-positive bacteria

PAPOUŠKOVÁ, Veronika, Pavel KADEŘÁVEK, Jiří NOVÁČEK, Petr PADRTA, Šanderová HANA, Rabatinová ALŽBĚTA, Zawadzka-Kazimierczuk ANNA, Kazimierczuk KRZYSZTOF, Lukáš ŽÍDEK, Kozminski WIKTOR, Krásný LIBOR and Vladimír SKLENÁŘ. Study of RNA polymerase delta subunit unique for gram-positive bacteria. In 9th Discussions in Structural Molecular Biology Nove Hrady. 2011. ISSN 1211-5894.

Basic information

• Original name: Study of RNA polymerase delta subunit unique for gram-positive bacteria
• Name in Czech: Studie delta podjednotky RNA polymerazi jedinečné pro gram-poziotivní bakterie
• Name (in English): Study of RNA polymerase delta subunit unique for gram-positive bacteria
• Authors: PAPOUŠKOVÁ, Veronika, Pavel KADEŘÁVEK, Jiří NOVÁČEK, Petr PADRTA, Šanderová HANA, Rabatinová ALŽBĚTA, Zawadzka-Kazimierczuk ANNA, Kazimierczuk KRZYSZTOF, Lukáš ŽÍDEK, Kozminski WIKTOR, Krásný LIBOR and Vladimír SKLENÁŘ.
• Edition: 9th Discussions in Structural Molecular Biology Nove Hrady, 2011.

Other information

• Type of outcome: Conference abstract
• Confidentiality degree: is not subject to a state or trade secret
• WWW: URL
• Organization unit: Faculty of Science
• ISSN: 1211-5894
• Keywords (in Czech): nmr, delta podjednotka, RNA polymeraza, struktura, dynamika, interakce
• Keywords in English: nmr, delta subunit, RNA polymerase, structure, dynamics, interactions

Abstract

RNA polymerase (RNAP) from gram-positive bacteria such as Bacillus subtilis differs from well-studied RNA polymerase from gram-negative bacteria in the presence of two additional subunits interacting with the core enzyme, delta and omega1. Recent results indicated that the presence of delta subunit increases the transcription specificity and the efficiency of RNA synthesis. Moreover, the absence of delta subunit is proposed to decrease a virulence of some pathogens. As crystallization at structure genomics centers failed, we focused on NMR studies of the delta subunit to reveal its structure and related dynamics. As the previous study showed (López de Saro et al., JMB, 1995), the C-terminal domain of the delta subunit is unstructured and its sequence is highly repetitive. Therefore, we started a systematic investigation of the protein with a shorten construct, corresponding to the well-structured N-terminal part. The construct constitution was confirmed using mass spectrometry and the secondary structure content as well as the protein thermostability were determined from circular dichroism spectra. So far, we have published its high-quality structure and the work on the analysis of internal motions is almost finished. The more challenging part of the protein, the C-terminal domain, was not initially studied because the NMR methodology for disordered, flexible proteins was not sufficiently developed at the beginning of the project. Fortunately, during last few years many new approaches for study of biologically interesting intrinsically disordered proteins appeared. In contrast to X-ray crystallography, NMR can provide valuable information on residual secondary structure, possible long-range contacts, and internal dynamics of the disordered polypeptide chain. The full-length delta protein was prepared using a standard protocol of overexpression in the E.coli system to produce a 15N,13C-uniformly labeled sample. The basic spectra, including a standard set of triple resonance NMR experiments, 3D TOCSY, and 3D NOESY, were measured on a 600MHz spectrometer. However, the analysis of the spectra was almost impossible due to a very small differences in chemical shifts. Therefore, a new methodology coming from Wiktor Ko¿mi¿ski lab was used to improve the spectra resolution and the full-size protein was then completely assigned. It was a major step for further analysis including secondary structure prediction, study of internal motions or measurement of dipolar couplings. The interactions between the delta subunit and the RNAP was studied by NMR titration and gel-shift assay to indicate which subunits are essential for binding of the protein to the core enzyme. The experience retrieved in the presented study will be used for the innovation of the seminar C9531 taught at Masaryk University.

Abstract (in English)

RNA polymerase (RNAP) from gram-positive bacteria such as Bacillus subtilis differs from well-studied RNA polymerase from gram-negative bacteria in the presence of two additional subunits interacting with the core enzyme, delta and omega1. Recent results indicated that the presence of delta subunit increases the transcription specificity and the efficiency of RNA synthesis. Moreover, the absence of delta subunit is proposed to decrease a virulence of some pathogens. As crystallization at structure genomics centers failed, we focused on NMR studies of the delta subunit to reveal its structure and related dynamics. As the previous study showed (López de Saro et al., JMB, 1995), the C-terminal domain of the delta subunit is unstructured and its sequence is highly repetitive. Therefore, we started a systematic investigation of the protein with a shorten construct, corresponding to the well-structured N-terminal part. The construct constitution was confirmed using mass spectrometry and the secondary structure content as well as the protein thermostability were determined from circular dichroism spectra. So far, we have published its high-quality structure and the work on the analysis of internal motions is almost finished. The more challenging part of the protein, the C-terminal domain, was not initially studied because the NMR methodology for disordered, flexible proteins was not sufficiently developed at the beginning of the project. Fortunately, during last few years many new approaches for study of biologically interesting intrinsically disordered proteins appeared. In contrast to X-ray crystallography, NMR can provide valuable information on residual secondary structure, possible long-range contacts, and internal dynamics of the disordered polypeptide chain. The full-length delta protein was prepared using a standard protocol of overexpression in the E.coli system to produce a 15N,13C-uniformly labeled sample. The basic spectra, including a standard set of triple resonance NMR experiments, 3D TOCSY, and 3D NOESY, were measured on a 600MHz spectrometer. However, the analysis of the spectra was almost impossible due to a very small differences in chemical shifts. Therefore, a new methodology coming from Wiktor Ko¿mi¿ski lab was used to improve the spectra resolution and the full-size protein was then completely assigned. It was a major step for further analysis including secondary structure prediction, study of internal motions or measurement of dipolar couplings. The interactions between the delta subunit and the RNAP was studied by NMR titration and gel-shift assay to indicate which subunits are essential for binding of the protein to the core enzyme. The experience retrieved in the presented study will be used for the innovation of the seminar C9531 taught at Masaryk University.

Links

• FRVS/2066/2011, interní kód MU: Name: Inovace předmětu "Strukturní biochemie - seminář" formou virtuální laboratoře a e-learningových odpovědníků

Investor: Ministry of Education, Youth and Sports of the CR, G - Students' Creative Activity

• GA204/09/0583, research and development project: Name: Delta podjednotka RNA polymerázy z Gram pozitivních bakterií (Acronym: DELTA)

Investor: Czech Science Foundation

• GD301/09/H004, research and development project: Name: Molekulární a strukturní biologie vybraných cytostatik. Od mechanistických studií k chemoterapii rakoviny

Investor: Czech Science Foundation

• LC06030, research and development project: Name: Biomolekulární centrum

Investor: Ministry of Education, Youth and Sports of the CR, Biomolecular centre

• MSM0021622413, plan (intention): Name: Proteiny v metabolismu a při interakci organismů s prostředím

Investor: Ministry of Education, Youth and Sports of the CR, Proteins in metabolism and interaction of organisms with the environment