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Quantitative analysis of processive RNA degradation by the archaeal RNA exosome.

Hartung S, Niederberger T, Hartung M, Tresch A, Hopfner KP - Nucleic Acids Res. (2010)

Bottom Line: Markov Chain Monte Carlo methods for parameter estimation allow for the comparison of reaction kinetics between different exosome variants and substrates.We show that long substrates are degraded in a processive and short RNA in a more distributive manner and that the cap proteins influence degradation speed.Our results, supported by small angle X-ray scattering, suggest that the Rrp4-type cap efficiently recruits RNA but prevents fast RNA degradation of longer RNAs by molecular friction, likely by RNA contacts to its unique KH-domain.

View Article: PubMed Central - PubMed

Affiliation: Center for Integrated Protein Sciences, Department of Biochemistry, Ludwig-Maximilians-University Munich, Munich, Germany.

ABSTRACT
RNA exosomes are large multisubunit assemblies involved in controlled RNA processing. The archaeal exosome possesses a heterohexameric processing chamber with three RNase-PH-like active sites, capped by Rrp4- or Csl4-type subunits containing RNA-binding domains. RNA degradation by RNA exosomes has not been studied in a quantitative manner because of the complex kinetics involved, and exosome features contributing to efficient RNA degradation remain unclear. Here we derive a quantitative kinetic model for degradation of a model substrate by the archaeal exosome. Markov Chain Monte Carlo methods for parameter estimation allow for the comparison of reaction kinetics between different exosome variants and substrates. We show that long substrates are degraded in a processive and short RNA in a more distributive manner and that the cap proteins influence degradation speed. Our results, supported by small angle X-ray scattering, suggest that the Rrp4-type cap efficiently recruits RNA but prevents fast RNA degradation of longer RNAs by molecular friction, likely by RNA contacts to its unique KH-domain. We also show that formation of the RNase-PH like ring with entrapped RNA is not required for high catalytic efficiency, suggesting that the exosome chamber evolved for controlled processivity, rather than for catalytic chemistry in RNA decay.

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Crystal structure of 6-mer RNA bound to the active site of the archaeal exosome. Rrp41 is shown in light and Rrp42 in dark green. The 2Fo–Fc electron density is contoured at 1.0σ and only shown for the RNA and the side chain of Y70Rrp41. (A) In the wild-type exosome Y70 is stacking with the fourth base of the bound RNA, and only weak density can be seen for the fifth and sixth base. (B) Electron density for the fourth base of the RNA is much weaker in the Y70ARrp41 mutant compared to the wild-type and no density can be detected at this contour level for additional nucleotides.
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Figure 2: Crystal structure of 6-mer RNA bound to the active site of the archaeal exosome. Rrp41 is shown in light and Rrp42 in dark green. The 2Fo–Fc electron density is contoured at 1.0σ and only shown for the RNA and the side chain of Y70Rrp41. (A) In the wild-type exosome Y70 is stacking with the fourth base of the bound RNA, and only weak density can be seen for the fifth and sixth base. (B) Electron density for the fourth base of the RNA is much weaker in the Y70ARrp41 mutant compared to the wild-type and no density can be detected at this contour level for additional nucleotides.

Mentions: Second, the final degradation product is a 3-mer. Further degradation is extremely slow, comparable to spontaneous background hydrolytic cleavage under the present conditions. We hypothesized that features of the active site might interact specifically with the fourth base at the 3′-end. Previous structural analysis with the Sulfolobus solfataricus exosome has shown that at least 4 nt are stably bound in the phosphoropytic active sites (26), but in the case of the Pyrococcus furiosus exosome some nucleotides were recognized (16). To get direct structural information for the A. fulgidus exosome:RNA interaction, used in this study, we crystallized our Csl4-exosome with a 6-mer RNA molecule (Figure 2; Supplementary Table S1). Four nucleotides from the 3′-end are clearly visible in the unbiased Fo–Fc electron density, with weaker density for the two additional nucleotides. Interestingly, the side chain of Y70Rrp42 shows π-stacking with the fourth base (counting from the active site) and this seems to be a conserved feature among archaeal exosomes (16,26). This interaction specifically stabilizes the first 4 nt, while RNA positions +5 and +6 behind Y70Rrp42 appear not to be specifically recognized. To test the role of Y70Rrp42, we determined the co-crystal structure of the Csl4-exosome-Y70A mutant with a CCCCUC oligonucleotide. In fact, we only see clear electron density for 4 nt in the active site and the electron density at position +4 is weaker and less defined compared to the wild-type. Thus, the 3-mer as degradation end-product is likely the cause of inefficient recognition of RNA’s with <4 nt at the active site.


Quantitative analysis of processive RNA degradation by the archaeal RNA exosome.

Hartung S, Niederberger T, Hartung M, Tresch A, Hopfner KP - Nucleic Acids Res. (2010)

Crystal structure of 6-mer RNA bound to the active site of the archaeal exosome. Rrp41 is shown in light and Rrp42 in dark green. The 2Fo–Fc electron density is contoured at 1.0σ and only shown for the RNA and the side chain of Y70Rrp41. (A) In the wild-type exosome Y70 is stacking with the fourth base of the bound RNA, and only weak density can be seen for the fifth and sixth base. (B) Electron density for the fourth base of the RNA is much weaker in the Y70ARrp41 mutant compared to the wild-type and no density can be detected at this contour level for additional nucleotides.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2926604&req=5

Figure 2: Crystal structure of 6-mer RNA bound to the active site of the archaeal exosome. Rrp41 is shown in light and Rrp42 in dark green. The 2Fo–Fc electron density is contoured at 1.0σ and only shown for the RNA and the side chain of Y70Rrp41. (A) In the wild-type exosome Y70 is stacking with the fourth base of the bound RNA, and only weak density can be seen for the fifth and sixth base. (B) Electron density for the fourth base of the RNA is much weaker in the Y70ARrp41 mutant compared to the wild-type and no density can be detected at this contour level for additional nucleotides.
Mentions: Second, the final degradation product is a 3-mer. Further degradation is extremely slow, comparable to spontaneous background hydrolytic cleavage under the present conditions. We hypothesized that features of the active site might interact specifically with the fourth base at the 3′-end. Previous structural analysis with the Sulfolobus solfataricus exosome has shown that at least 4 nt are stably bound in the phosphoropytic active sites (26), but in the case of the Pyrococcus furiosus exosome some nucleotides were recognized (16). To get direct structural information for the A. fulgidus exosome:RNA interaction, used in this study, we crystallized our Csl4-exosome with a 6-mer RNA molecule (Figure 2; Supplementary Table S1). Four nucleotides from the 3′-end are clearly visible in the unbiased Fo–Fc electron density, with weaker density for the two additional nucleotides. Interestingly, the side chain of Y70Rrp42 shows π-stacking with the fourth base (counting from the active site) and this seems to be a conserved feature among archaeal exosomes (16,26). This interaction specifically stabilizes the first 4 nt, while RNA positions +5 and +6 behind Y70Rrp42 appear not to be specifically recognized. To test the role of Y70Rrp42, we determined the co-crystal structure of the Csl4-exosome-Y70A mutant with a CCCCUC oligonucleotide. In fact, we only see clear electron density for 4 nt in the active site and the electron density at position +4 is weaker and less defined compared to the wild-type. Thus, the 3-mer as degradation end-product is likely the cause of inefficient recognition of RNA’s with <4 nt at the active site.

Bottom Line: Markov Chain Monte Carlo methods for parameter estimation allow for the comparison of reaction kinetics between different exosome variants and substrates.We show that long substrates are degraded in a processive and short RNA in a more distributive manner and that the cap proteins influence degradation speed.Our results, supported by small angle X-ray scattering, suggest that the Rrp4-type cap efficiently recruits RNA but prevents fast RNA degradation of longer RNAs by molecular friction, likely by RNA contacts to its unique KH-domain.

View Article: PubMed Central - PubMed

Affiliation: Center for Integrated Protein Sciences, Department of Biochemistry, Ludwig-Maximilians-University Munich, Munich, Germany.

ABSTRACT
RNA exosomes are large multisubunit assemblies involved in controlled RNA processing. The archaeal exosome possesses a heterohexameric processing chamber with three RNase-PH-like active sites, capped by Rrp4- or Csl4-type subunits containing RNA-binding domains. RNA degradation by RNA exosomes has not been studied in a quantitative manner because of the complex kinetics involved, and exosome features contributing to efficient RNA degradation remain unclear. Here we derive a quantitative kinetic model for degradation of a model substrate by the archaeal exosome. Markov Chain Monte Carlo methods for parameter estimation allow for the comparison of reaction kinetics between different exosome variants and substrates. We show that long substrates are degraded in a processive and short RNA in a more distributive manner and that the cap proteins influence degradation speed. Our results, supported by small angle X-ray scattering, suggest that the Rrp4-type cap efficiently recruits RNA but prevents fast RNA degradation of longer RNAs by molecular friction, likely by RNA contacts to its unique KH-domain. We also show that formation of the RNase-PH like ring with entrapped RNA is not required for high catalytic efficiency, suggesting that the exosome chamber evolved for controlled processivity, rather than for catalytic chemistry in RNA decay.

Show MeSH
Related in: MedlinePlus