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A non-canonical multisubunit RNA polymerase encoded by a giant bacteriophage.

Yakunina M, Artamonova T, Borukhov S, Makarova KS, Severinov K, Minakhin L - Nucleic Acids Res. (2015)

Bottom Line: Polypeptides of one set are encoded by middle phage genes and are found in the phiKZ virions.Thus, this enzyme is a non-virion phiKZ RNAP responsible for transcription of late phage genes.The phiKZ RNAP lacks identifiable assembly and promoter specificity subunits/factors characteristic for eukaryal, archaeal and bacterial RNAPs and thus provides a unique model for comparative analysis of the mechanism, regulation and evolution of this important class of enzymes.

View Article: PubMed Central - PubMed

Affiliation: Peter the Great St. Petersburg Polytechnic University, St. Petersburg, 195251, Russia Department of Molecular Biology and Biochemistry, Waksman Institute, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020, USA.

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Purification of phiKZ-encoded nvRNAP. (A) The sequence of steps used to purify nvRNAP is shown. (B) А Coomassie-stained SDS gel of fractions containing gp55 and gp74 during nvRNAP purification with the gel lane numbers corresponding to the numbers of steps from panel А. (C) Native PAGE analysis of nvRNAP from the final MonoQ purification step (upper panel) and subunit composition of the native PAGE band (lower panel). Individual protein bands were identified by mass-spectrometry.
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Figure 1: Purification of phiKZ-encoded nvRNAP. (A) The sequence of steps used to purify nvRNAP is shown. (B) А Coomassie-stained SDS gel of fractions containing gp55 and gp74 during nvRNAP purification with the gel lane numbers corresponding to the numbers of steps from panel А. (C) Native PAGE analysis of nvRNAP from the final MonoQ purification step (upper panel) and subunit composition of the native PAGE band (lower panel). Individual protein bands were identified by mass-spectrometry.

Mentions: Purification of phage RNAPs from phiKZ-infected P. aeruginosa cells was undertaken. Cells were collected 30 min post infection, at the onset of late phage transcription (30). The procedure was based on the standard purification of bacterial RNAP (36). The presence of phage RNAP-like proteins was monitored by mass-spectrometric analysis of protein bands after SDS-PAGE separation of components of individual fractions. After polyethylenimine P precipitation, a standard first step during bacterial RNAP purification (36), an extract of the pellet with a buffer containing 0.3 M NaCl was found to contain non-virion RNAP-like proteins gp55 and gp74. Host RNAP β and β’ and proteins of the putative virion phage RNAP-like were not detected in this extract. We decided to concentrate on this sample with a goal of purifying gp55, gp74 and any associated proteins. A sequence of chromatographic steps (including heparin-sepharose, DEAE-sepharose, Superdex 200 and MonoQ columns) was elaborated with electrophoretic and mass-spectrometric monitoring of gp55 and gp74 in the fractions (Figure 1A). As a result, a single peak eluting from a MonoQ column was obtained. SDS-PAGE revealed the presence of five polypeptides in apparently stoichiometric amounts in peak fractions (Figure 1B, lane 6). Native PAGE of the material from peak fraction revealed a single band. SDS-PAGE analysis of polypeptide content of the native gel band revealed all five polypeptides (Figure 1C), which, therefore, form a stable complex. Mass-spectrometric analysis demonstrated that the complex consists of the five polypeptides: gp55 (56 kDa), gp68 (59 kDa), gp71–73 (74 kDa), gp74 (77 kDa) and gp123 (63 kDa), hereafter referred to as phiKZ non-virion (nv) RNAP.


A non-canonical multisubunit RNA polymerase encoded by a giant bacteriophage.

Yakunina M, Artamonova T, Borukhov S, Makarova KS, Severinov K, Minakhin L - Nucleic Acids Res. (2015)

Purification of phiKZ-encoded nvRNAP. (A) The sequence of steps used to purify nvRNAP is shown. (B) А Coomassie-stained SDS gel of fractions containing gp55 and gp74 during nvRNAP purification with the gel lane numbers corresponding to the numbers of steps from panel А. (C) Native PAGE analysis of nvRNAP from the final MonoQ purification step (upper panel) and subunit composition of the native PAGE band (lower panel). Individual protein bands were identified by mass-spectrometry.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4666361&req=5

Figure 1: Purification of phiKZ-encoded nvRNAP. (A) The sequence of steps used to purify nvRNAP is shown. (B) А Coomassie-stained SDS gel of fractions containing gp55 and gp74 during nvRNAP purification with the gel lane numbers corresponding to the numbers of steps from panel А. (C) Native PAGE analysis of nvRNAP from the final MonoQ purification step (upper panel) and subunit composition of the native PAGE band (lower panel). Individual protein bands were identified by mass-spectrometry.
Mentions: Purification of phage RNAPs from phiKZ-infected P. aeruginosa cells was undertaken. Cells were collected 30 min post infection, at the onset of late phage transcription (30). The procedure was based on the standard purification of bacterial RNAP (36). The presence of phage RNAP-like proteins was monitored by mass-spectrometric analysis of protein bands after SDS-PAGE separation of components of individual fractions. After polyethylenimine P precipitation, a standard first step during bacterial RNAP purification (36), an extract of the pellet with a buffer containing 0.3 M NaCl was found to contain non-virion RNAP-like proteins gp55 and gp74. Host RNAP β and β’ and proteins of the putative virion phage RNAP-like were not detected in this extract. We decided to concentrate on this sample with a goal of purifying gp55, gp74 and any associated proteins. A sequence of chromatographic steps (including heparin-sepharose, DEAE-sepharose, Superdex 200 and MonoQ columns) was elaborated with electrophoretic and mass-spectrometric monitoring of gp55 and gp74 in the fractions (Figure 1A). As a result, a single peak eluting from a MonoQ column was obtained. SDS-PAGE revealed the presence of five polypeptides in apparently stoichiometric amounts in peak fractions (Figure 1B, lane 6). Native PAGE of the material from peak fraction revealed a single band. SDS-PAGE analysis of polypeptide content of the native gel band revealed all five polypeptides (Figure 1C), which, therefore, form a stable complex. Mass-spectrometric analysis demonstrated that the complex consists of the five polypeptides: gp55 (56 kDa), gp68 (59 kDa), gp71–73 (74 kDa), gp74 (77 kDa) and gp123 (63 kDa), hereafter referred to as phiKZ non-virion (nv) RNAP.

Bottom Line: Polypeptides of one set are encoded by middle phage genes and are found in the phiKZ virions.Thus, this enzyme is a non-virion phiKZ RNAP responsible for transcription of late phage genes.The phiKZ RNAP lacks identifiable assembly and promoter specificity subunits/factors characteristic for eukaryal, archaeal and bacterial RNAPs and thus provides a unique model for comparative analysis of the mechanism, regulation and evolution of this important class of enzymes.

View Article: PubMed Central - PubMed

Affiliation: Peter the Great St. Petersburg Polytechnic University, St. Petersburg, 195251, Russia Department of Molecular Biology and Biochemistry, Waksman Institute, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020, USA.

Show MeSH
Related in: MedlinePlus