Limits...
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.

Show MeSH

Related in: MedlinePlus

Visualization of RNase activity of the archaeal exosome on denaturing polyacrylamide gels: the input (I) is a 30-mer polyA RNA radioactively labelled at the 5′-end that is degraded from the 3′-end to a final product (FP) of a 3-mer. Time points were taken in increasing intervals [in minutes: 0:10; 0:20; 0:30; 0:40; 0:50; 1:00; 1:10; 1:20; 1:40; 2:00; 2:20; 2:40; 3:00; 3:30; 4:00; 4:30; 5:00; 5:50; 6:00; 6:30; 7:00; 7:30; 8:00; 9:00; 10:00; 12:00; 14:00; 16:00; 18:00; 20:00; 25:00; 30:00; 35:00; 40:00; (B) ends at 8:00 min]. RNA degradation does clearly not occur with constant speed and the (Csl4:Rrp41:Rrp42)3 exosome (A) degrades RNA with a different time dependency than the (Rrp4:Rrp41:Rrp42)3 exosome (B).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2926604&req=5

Figure 1: Visualization of RNase activity of the archaeal exosome on denaturing polyacrylamide gels: the input (I) is a 30-mer polyA RNA radioactively labelled at the 5′-end that is degraded from the 3′-end to a final product (FP) of a 3-mer. Time points were taken in increasing intervals [in minutes: 0:10; 0:20; 0:30; 0:40; 0:50; 1:00; 1:10; 1:20; 1:40; 2:00; 2:20; 2:40; 3:00; 3:30; 4:00; 4:30; 5:00; 5:50; 6:00; 6:30; 7:00; 7:30; 8:00; 9:00; 10:00; 12:00; 14:00; 16:00; 18:00; 20:00; 25:00; 30:00; 35:00; 40:00; (B) ends at 8:00 min]. RNA degradation does clearly not occur with constant speed and the (Csl4:Rrp41:Rrp42)3 exosome (A) degrades RNA with a different time dependency than the (Rrp4:Rrp41:Rrp42)3 exosome (B).

Mentions: Three different models to describe the kinetics of RNA degradation by the exosome were tested: (A) scheme for the general kinetic model, which includes cleavage and polymerization rates kc and kp as well as association and dissociation rates ka and kd for all RNAs from 30–4 nt. (B–D) Quantified concentrations of RNA intermediates from Figure 1A, along with least square fits to different kinetic models. (B) Strict processivity considers only 27 different cleavage rates kc,30–kc,4. (C) cleavage-and-polymerization considers 27 different cleavage rates kc,30–kc,4, 27 different polymerization rates kp,30–kp,4 and one initial association rate ka,30 (=55 rates). With models (C) and (B), no reasonable fit could be obtained. (D) By including association, dissociation and cleavage and making rational simplifications (see text) we can convincingly fit the data with a model containing 28 different rate constants.


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)

Visualization of RNase activity of the archaeal exosome on denaturing polyacrylamide gels: the input (I) is a 30-mer polyA RNA radioactively labelled at the 5′-end that is degraded from the 3′-end to a final product (FP) of a 3-mer. Time points were taken in increasing intervals [in minutes: 0:10; 0:20; 0:30; 0:40; 0:50; 1:00; 1:10; 1:20; 1:40; 2:00; 2:20; 2:40; 3:00; 3:30; 4:00; 4:30; 5:00; 5:50; 6:00; 6:30; 7:00; 7:30; 8:00; 9:00; 10:00; 12:00; 14:00; 16:00; 18:00; 20:00; 25:00; 30:00; 35:00; 40:00; (B) ends at 8:00 min]. RNA degradation does clearly not occur with constant speed and the (Csl4:Rrp41:Rrp42)3 exosome (A) degrades RNA with a different time dependency than the (Rrp4:Rrp41:Rrp42)3 exosome (B).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Visualization of RNase activity of the archaeal exosome on denaturing polyacrylamide gels: the input (I) is a 30-mer polyA RNA radioactively labelled at the 5′-end that is degraded from the 3′-end to a final product (FP) of a 3-mer. Time points were taken in increasing intervals [in minutes: 0:10; 0:20; 0:30; 0:40; 0:50; 1:00; 1:10; 1:20; 1:40; 2:00; 2:20; 2:40; 3:00; 3:30; 4:00; 4:30; 5:00; 5:50; 6:00; 6:30; 7:00; 7:30; 8:00; 9:00; 10:00; 12:00; 14:00; 16:00; 18:00; 20:00; 25:00; 30:00; 35:00; 40:00; (B) ends at 8:00 min]. RNA degradation does clearly not occur with constant speed and the (Csl4:Rrp41:Rrp42)3 exosome (A) degrades RNA with a different time dependency than the (Rrp4:Rrp41:Rrp42)3 exosome (B).
Mentions: Three different models to describe the kinetics of RNA degradation by the exosome were tested: (A) scheme for the general kinetic model, which includes cleavage and polymerization rates kc and kp as well as association and dissociation rates ka and kd for all RNAs from 30–4 nt. (B–D) Quantified concentrations of RNA intermediates from Figure 1A, along with least square fits to different kinetic models. (B) Strict processivity considers only 27 different cleavage rates kc,30–kc,4. (C) cleavage-and-polymerization considers 27 different cleavage rates kc,30–kc,4, 27 different polymerization rates kp,30–kp,4 and one initial association rate ka,30 (=55 rates). With models (C) and (B), no reasonable fit could be obtained. (D) By including association, dissociation and cleavage and making rational simplifications (see text) we can convincingly fit the data with a model containing 28 different rate constants.

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