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An RNA degradosome assembly in Caulobacter crescentus.

Hardwick SW, Chan VS, Broadhurst RW, Luisi BF - Nucleic Acids Res. (2010)

Bottom Line: These recognition 'microdomains' punctuate structurally an extensive region that is otherwise predicted to be natively disordered.Finally, we observe that the abundance of RNase E varies through the cell cycle, with maxima at morphological differentiation and cell division.This variation may contribute to the program of gene expression during cell division.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.

ABSTRACT
In many bacterial species, the multi-enzyme RNA degradosome assembly makes key contributions to RNA metabolism. Powering the turnover of RNA and the processing of structural precursors, the RNA degradosome has differential activities on a spectrum of transcripts and contributes to gene regulation at a global level. Here, we report the isolation and characterization of an RNA degradosome assembly from the α-proteobacterium Caulobacter crescentus, which is a model organism for studying morphological development and cell-cycle progression. The principal components of the C. crescentus degradosome are the endoribonuclease RNase E, the exoribonuclease polynucleotide phosphorylase (PNPase), a DEAD-box RNA helicase and the Krebs cycle enzyme aconitase. PNPase and aconitase associate with specific segments in the C-terminal domain of RNase E that are predicted to have structural propensity. These recognition 'microdomains' punctuate structurally an extensive region that is otherwise predicted to be natively disordered. Finally, we observe that the abundance of RNase E varies through the cell cycle, with maxima at morphological differentiation and cell division. This variation may contribute to the program of gene expression during cell division.

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RNase E of α-proteobacteria have a distinguishing S1 domain insert not found in RNase E of other bacterial classes. (A) Structure based sequence alignment of the E. coli and C. crescentus RNase E catalytic domains. The secondary structural elements of E. coli RNase E are shown on the lines above the sequence alignment using the PDB file 2BX2. The arrows indicate β-sheet, the coils indicate α-helices, TT indicates β turns and η indicates 310 helices. Red letters indicate homology and blue boxes show similarity. The red highlights indicate identity across the sequences. The green stars represent the antigenic peptide used in this study. Structural sub-domains of RNase E are coloured (RNase H: light blue; S1: purple; 5′ sensor: yellow; DNase I: dark grey; zinc link: red; small domain: dark blue). Alignments were prepared using CLUSTALW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html) and ESPript (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). (B) E. coli RNase E NTD tetramer in complex with 13-mer RNA (pale orange). Structural sub-domains are highlighted for one protomer, coloured as in Figure 1A. The position of the S1 insert absent in E. coli RNase E catalytic domain is represented by the dashed loops.
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Figure 1: RNase E of α-proteobacteria have a distinguishing S1 domain insert not found in RNase E of other bacterial classes. (A) Structure based sequence alignment of the E. coli and C. crescentus RNase E catalytic domains. The secondary structural elements of E. coli RNase E are shown on the lines above the sequence alignment using the PDB file 2BX2. The arrows indicate β-sheet, the coils indicate α-helices, TT indicates β turns and η indicates 310 helices. Red letters indicate homology and blue boxes show similarity. The red highlights indicate identity across the sequences. The green stars represent the antigenic peptide used in this study. Structural sub-domains of RNase E are coloured (RNase H: light blue; S1: purple; 5′ sensor: yellow; DNase I: dark grey; zinc link: red; small domain: dark blue). Alignments were prepared using CLUSTALW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html) and ESPript (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). (B) E. coli RNase E NTD tetramer in complex with 13-mer RNA (pale orange). Structural sub-domains are highlighted for one protomer, coloured as in Figure 1A. The position of the S1 insert absent in E. coli RNase E catalytic domain is represented by the dashed loops.

Mentions: A rabbit polyclonal antibody was raised against a 16-mer peptide (RDDSGDDEDDTPIRSRR) corresponding to a segment of the S1 subdomain of the C. crescentus RNase E catalytic domain. The antigenic peptide is part of a poorly conserved insert in α-proteobacteria that is absent in RNase E of other bacterial classes (Figure 1A), although it does occur in plant homologues (34). We suspected that the S1 insert region would provide a suitable antibody binding epitope as this moiety is highly charged and, according to the PONDR disorder prediction algorithm (35,36), is intrinsically unstructured (Figure 4A). The crystal structure of the E. coli RNase E catalytic domain (37) suggests that the S1 subdomain should be exposed and accessible (Figure 1B).Figure 1.


An RNA degradosome assembly in Caulobacter crescentus.

Hardwick SW, Chan VS, Broadhurst RW, Luisi BF - Nucleic Acids Res. (2010)

RNase E of α-proteobacteria have a distinguishing S1 domain insert not found in RNase E of other bacterial classes. (A) Structure based sequence alignment of the E. coli and C. crescentus RNase E catalytic domains. The secondary structural elements of E. coli RNase E are shown on the lines above the sequence alignment using the PDB file 2BX2. The arrows indicate β-sheet, the coils indicate α-helices, TT indicates β turns and η indicates 310 helices. Red letters indicate homology and blue boxes show similarity. The red highlights indicate identity across the sequences. The green stars represent the antigenic peptide used in this study. Structural sub-domains of RNase E are coloured (RNase H: light blue; S1: purple; 5′ sensor: yellow; DNase I: dark grey; zinc link: red; small domain: dark blue). Alignments were prepared using CLUSTALW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html) and ESPript (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). (B) E. coli RNase E NTD tetramer in complex with 13-mer RNA (pale orange). Structural sub-domains are highlighted for one protomer, coloured as in Figure 1A. The position of the S1 insert absent in E. coli RNase E catalytic domain is represented by the dashed loops.
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Related In: Results  -  Collection

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Figure 1: RNase E of α-proteobacteria have a distinguishing S1 domain insert not found in RNase E of other bacterial classes. (A) Structure based sequence alignment of the E. coli and C. crescentus RNase E catalytic domains. The secondary structural elements of E. coli RNase E are shown on the lines above the sequence alignment using the PDB file 2BX2. The arrows indicate β-sheet, the coils indicate α-helices, TT indicates β turns and η indicates 310 helices. Red letters indicate homology and blue boxes show similarity. The red highlights indicate identity across the sequences. The green stars represent the antigenic peptide used in this study. Structural sub-domains of RNase E are coloured (RNase H: light blue; S1: purple; 5′ sensor: yellow; DNase I: dark grey; zinc link: red; small domain: dark blue). Alignments were prepared using CLUSTALW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html) and ESPript (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). (B) E. coli RNase E NTD tetramer in complex with 13-mer RNA (pale orange). Structural sub-domains are highlighted for one protomer, coloured as in Figure 1A. The position of the S1 insert absent in E. coli RNase E catalytic domain is represented by the dashed loops.
Mentions: A rabbit polyclonal antibody was raised against a 16-mer peptide (RDDSGDDEDDTPIRSRR) corresponding to a segment of the S1 subdomain of the C. crescentus RNase E catalytic domain. The antigenic peptide is part of a poorly conserved insert in α-proteobacteria that is absent in RNase E of other bacterial classes (Figure 1A), although it does occur in plant homologues (34). We suspected that the S1 insert region would provide a suitable antibody binding epitope as this moiety is highly charged and, according to the PONDR disorder prediction algorithm (35,36), is intrinsically unstructured (Figure 4A). The crystal structure of the E. coli RNase E catalytic domain (37) suggests that the S1 subdomain should be exposed and accessible (Figure 1B).Figure 1.

Bottom Line: These recognition 'microdomains' punctuate structurally an extensive region that is otherwise predicted to be natively disordered.Finally, we observe that the abundance of RNase E varies through the cell cycle, with maxima at morphological differentiation and cell division.This variation may contribute to the program of gene expression during cell division.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.

ABSTRACT
In many bacterial species, the multi-enzyme RNA degradosome assembly makes key contributions to RNA metabolism. Powering the turnover of RNA and the processing of structural precursors, the RNA degradosome has differential activities on a spectrum of transcripts and contributes to gene regulation at a global level. Here, we report the isolation and characterization of an RNA degradosome assembly from the α-proteobacterium Caulobacter crescentus, which is a model organism for studying morphological development and cell-cycle progression. The principal components of the C. crescentus degradosome are the endoribonuclease RNase E, the exoribonuclease polynucleotide phosphorylase (PNPase), a DEAD-box RNA helicase and the Krebs cycle enzyme aconitase. PNPase and aconitase associate with specific segments in the C-terminal domain of RNase E that are predicted to have structural propensity. These recognition 'microdomains' punctuate structurally an extensive region that is otherwise predicted to be natively disordered. Finally, we observe that the abundance of RNase E varies through the cell cycle, with maxima at morphological differentiation and cell division. This variation may contribute to the program of gene expression during cell division.

Show MeSH
Related in: MedlinePlus