Limits...
Structural and biochemical investigation of bacteriophage N4-encoded RNA polymerases.

Lenneman BR, Rothman-Denes LB - Biomolecules (2015)

Bottom Line: These encode the components of new transcriptional machinery (N4 RNAPII and cofactors) responsible for the synthesis of middle RNAs.Both N4 RNAPs belong to the T7-like "single-subunit" family of polymerases.Herein, we describe their mechanisms of promoter recognition, regulation, and roles in the phage life cycle.

View Article: PubMed Central - PubMed

Affiliation: Committee on Genetics, Genomics, and Systems Biology, The University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA. blenneman@uchicago.edu.

ABSTRACT
Bacteriophage N4 regulates the temporal expression of its genome through the activity of three distinct RNA polymerases (RNAP). Expression of the early genes is carried out by a phage-encoded, virion-encapsidated RNAP (vRNAP) that is injected into the host at the onset of infection and transcribes the early genes. These encode the components of new transcriptional machinery (N4 RNAPII and cofactors) responsible for the synthesis of middle RNAs. Both N4 RNAPs belong to the T7-like "single-subunit" family of polymerases. Herein, we describe their mechanisms of promoter recognition, regulation, and roles in the phage life cycle.

Show MeSH

Related in: MedlinePlus

Model of middle transcription by N4 RNAPII. AT-rich double-stranded promoters are specifically recognized and melted by an unknown protein “X,” allowing gp2 to bind to single-stranded DNA. Gp2 recruits N4 RNAPII to the promoter through direct interactions, allowing N4 RNAPII to recognize specific sequences in the template strand and initiate transcription. Template and non-template strands of promoter DNA are represented as purple and pink, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4496689&req=5

biomolecules-05-00647-f007: Model of middle transcription by N4 RNAPII. AT-rich double-stranded promoters are specifically recognized and melted by an unknown protein “X,” allowing gp2 to bind to single-stranded DNA. Gp2 recruits N4 RNAPII to the promoter through direct interactions, allowing N4 RNAPII to recognize specific sequences in the template strand and initiate transcription. Template and non-template strands of promoter DNA are represented as purple and pink, respectively.

Mentions: To elucidate the role of gp2 in middle transcription, ORF2 was cloned and sequenced, revealing no homology to proteins of known function [57]. The purified protein was tested for DNA binding and transcription-enhancing properties in vitro. Surprisingly, gp2 was shown to be a single-stranded DNA-binding protein exhibiting no sequence specificity [57]. It activates transcription through a recruitment mechanism, as binding of gp2 to single-stranded DNA increased the affinity of N4 RNAPII for the same template. Recruitment occurs through binding to N4 RNAPII; the N4 RNAPII-gp2 complex withstands treatment with high salt [57]. The data presented above has led us to propose a model for middle transcription whereby an unknown protein binds to double-stranded DNA to unwind the AT-rich promoter sequence, allowing gp2 to bind to the single-stranded DNA at middle promoters and recruit N4 RNAPII through direct interactions to the promoter sequences. N4 RNAPII then recognizes specific sequences in the template strand and initiates transcription (Figure 7). Preliminary evidence suggests that the product of ORF1, gp1, is required for middle promoter utilization in vivo. Experiments to characterize gp1’s role in middle transcription and define the contacts required for N4 RNAPII promoter recognition are currently in progress.


Structural and biochemical investigation of bacteriophage N4-encoded RNA polymerases.

Lenneman BR, Rothman-Denes LB - Biomolecules (2015)

Model of middle transcription by N4 RNAPII. AT-rich double-stranded promoters are specifically recognized and melted by an unknown protein “X,” allowing gp2 to bind to single-stranded DNA. Gp2 recruits N4 RNAPII to the promoter through direct interactions, allowing N4 RNAPII to recognize specific sequences in the template strand and initiate transcription. Template and non-template strands of promoter DNA are represented as purple and pink, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496689&req=5

biomolecules-05-00647-f007: Model of middle transcription by N4 RNAPII. AT-rich double-stranded promoters are specifically recognized and melted by an unknown protein “X,” allowing gp2 to bind to single-stranded DNA. Gp2 recruits N4 RNAPII to the promoter through direct interactions, allowing N4 RNAPII to recognize specific sequences in the template strand and initiate transcription. Template and non-template strands of promoter DNA are represented as purple and pink, respectively.
Mentions: To elucidate the role of gp2 in middle transcription, ORF2 was cloned and sequenced, revealing no homology to proteins of known function [57]. The purified protein was tested for DNA binding and transcription-enhancing properties in vitro. Surprisingly, gp2 was shown to be a single-stranded DNA-binding protein exhibiting no sequence specificity [57]. It activates transcription through a recruitment mechanism, as binding of gp2 to single-stranded DNA increased the affinity of N4 RNAPII for the same template. Recruitment occurs through binding to N4 RNAPII; the N4 RNAPII-gp2 complex withstands treatment with high salt [57]. The data presented above has led us to propose a model for middle transcription whereby an unknown protein binds to double-stranded DNA to unwind the AT-rich promoter sequence, allowing gp2 to bind to the single-stranded DNA at middle promoters and recruit N4 RNAPII through direct interactions to the promoter sequences. N4 RNAPII then recognizes specific sequences in the template strand and initiates transcription (Figure 7). Preliminary evidence suggests that the product of ORF1, gp1, is required for middle promoter utilization in vivo. Experiments to characterize gp1’s role in middle transcription and define the contacts required for N4 RNAPII promoter recognition are currently in progress.

Bottom Line: These encode the components of new transcriptional machinery (N4 RNAPII and cofactors) responsible for the synthesis of middle RNAs.Both N4 RNAPs belong to the T7-like "single-subunit" family of polymerases.Herein, we describe their mechanisms of promoter recognition, regulation, and roles in the phage life cycle.

View Article: PubMed Central - PubMed

Affiliation: Committee on Genetics, Genomics, and Systems Biology, The University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA. blenneman@uchicago.edu.

ABSTRACT
Bacteriophage N4 regulates the temporal expression of its genome through the activity of three distinct RNA polymerases (RNAP). Expression of the early genes is carried out by a phage-encoded, virion-encapsidated RNAP (vRNAP) that is injected into the host at the onset of infection and transcribes the early genes. These encode the components of new transcriptional machinery (N4 RNAPII and cofactors) responsible for the synthesis of middle RNAs. Both N4 RNAPs belong to the T7-like "single-subunit" family of polymerases. Herein, we describe their mechanisms of promoter recognition, regulation, and roles in the phage life cycle.

Show MeSH
Related in: MedlinePlus