Linkage between Substrate Recognition and Catalysis during Cleavage of Sarcin/Ricin Loop RNA by Restrictocin

Korennykh, Alexei; Plantinga, Matthew J.; Correll, Carl C.; Piccirilli, Joseph A.

doi:10.1021/bi700931y

Cited by 18 publications

(61 citation statements)

References 33 publications

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“…The four residues and nucleotides A4318 and G4319 (shown in red) make the largest contributions to the difference between ⌬G el*(rib trun) and ⌬Gel*(SRL). phate is replaced with sulfur, allowing restrictocin to reach the diffusion-controlled limit (2). The experimental value of k cat /K m for the substituted substrate is very close to our calculated value of k a and, as expected from theory, is inversely proportion to solution viscosity.…”

Section: Resultssupporting

confidence: 87%

“…2, k a can be obtained from k cat /K m if k d and k cat are known. The data of Korennykh et al (2) indicate k d Ϸ8 s Ϫ1 and k cat Ϸ1 s Ϫ1 for restrictocin cleaving the SRL RNA at I ϭ 15 mM. Given that ionic strength has similar effects on k a and K a , the dissociation constant k d is expected to be independent of ionic strength.…”

Section: Resultsmentioning

confidence: 98%

“…The cleavage disrupts the binding of elongation factors to the ribosome, thereby halting protein synthesis and triggering apoptosis. Besides the sequence specificity of the substrate, another remarkable feature of ribotoxins is the record-setting rate constant, exceeding 10 10 M Ϫ1 s Ϫ1 at low ionic strengths, for binding the ribosome target (1,2). These two features are shared to a large extent by a related family, represented by ricin, known as ribosome-inactivating proteins (3).…”

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confidence: 99%

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Dissection of the high rate constant for the binding of a ribotoxin to the ribosome

Qin

Zhou

2009

Proc. Natl. Acad. Sci. U.S.A.

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Restrictocin belongs to a family of site-specific ribonucleases that kill cells by inactivating the ribosome. The restrictocin–ribosome binding rate constant was observed to exceed 1010 M−1 s−1. We have developed a transient-complex theory to model the binding rates of protein–protein and protein–RNA complexes. The theory predicts the rate constant as ka = ka0 exp(−ΔGel*/kBT), where ka0 is the basal rate constant for reaching the transient complex, located at the outer boundary of the bound state, by random diffusion, and ΔGel* is the average electrostatic interaction free energy of the transient complex. Here, we applied the transient-complex theory to dissect the high restrictocin–ribosome binding rate constant. We found that the binding rate of restrictocin to the isolated sarcin/ricin loop is electrostatically enhanced by ≈300-fold, similar to results found in other protein–protein and protein–RNA complexes. The ribosome provides an additional 10,000-fold rate enhancement because of two synergistic mechanisms afforded by the distal regions of the ribosome. First, they provide additional electrostatic attraction with restrictocin. Second, they reposition the transient complex into a region where local electrostatic interactions of restrictocin with the sarcin/ricin loop are particularly favorable. Our calculations rationalize a host of experimental observations and identify a strategy for designing proteins that bind their targets with high speed.

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Section: Resultssupporting

confidence: 87%

Section: Resultsmentioning

confidence: 98%

mentioning

confidence: 99%

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Dissection of the high rate constant for the binding of a ribotoxin to the ribosome

Qin

Zhou

2009

Proc. Natl. Acad. Sci. U.S.A.

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“…These results are not unexpected given the energetically frustrated nature of the RNA folding process, caused by RNA's relatively small number of chemical building blocks compared to that of proteins [9], [39]. Such frustrated folding leads to a rugged free energy landscape in vitro , featuring multiple local and global minima that can represent either functionally active or inactive states [12]–[16]. Our results suggest that segmental folding during transcription safely guides an RNA across the rugged folding free energy landscape towards the native state(s) in a way that heat annealing does not (we note that both the HDV and VS ribozyme have self-cleaved during transcription, attesting to the fact that they are both natively folded).…”

Section: Discussionmentioning

confidence: 90%

“…Central to most in vitro study has therefore been the (often implicit) assumption that RNA molecules properly refold into the native tertiary structures found in vivo , which are produced by co-transcriptional folding [8], often with the aid of folding enzymes called chaperones [7], [9]–[11]. Recently, evidence has accumulated for the existence of multiple, stable, functionally either active (native) or inactive (nonnative, misfolded) states of RNAs when refolded in vitro [12]–[16]. These results give renewed urgency to the still open question of whether refolding an entire RNA in vitro is a generally acceptable replacement for the segmental folding that occurs during transcription [7].…”

Section: Introductionmentioning

confidence: 99%

Nondenaturing Purification of Co-Transcriptionally Folded RNA Avoids Common Folding Heterogeneity

2010

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Due to the energetic frustration of RNA folding, tertiary structured RNA is typically characterized by a rugged folding free energy landscape where deep kinetic barriers separate numerous misfolded states from one or more native states. While most in vitro studies of RNA rely on (re)folding chemically and/or enzymatically synthesized RNA in its entirety, which frequently leads into kinetic traps, nature reduces the complexity of the RNA folding problem by segmental, co-transcriptional folding starting from the 5′ end. We here have developed a simplified, general, nondenaturing purification protocol for RNA to ask whether avoiding denaturation of a co-transcriptionally folded RNA can reduce commonly observed in vitro folding heterogeneity. Our protocol bypasses the need for large-scale auxiliary protein purification and expensive chromatographic equipment and involves rapid affinity capture with magnetic beads and removal of chemical heterogeneity by cleavage of the target RNA from the beads using the ligand-induced glmS ribozyme. For two disparate model systems, the Varkud satellite (VS) and hepatitis delta virus (HDV) ribozymes, we achieve >95% conformational purity within one hour of enzymatic transcription, without the need for any folding chaperones. We further demonstrate that in vitro refolding introduces severe conformational heterogeneity into the natively-purified VS ribozyme but not into the compact, double-nested pseudoknot fold of the HDV ribozyme. We conclude that conformational heterogeneity in complex RNAs can be avoided by co-transcriptional folding followed by nondenaturing purification, providing rapid access to chemically and conformationally pure RNA for biologically relevant biochemical and biophysical studies.

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RNAStructure: Tetraloops

Cheong

2010

Encyclopedia of Life Sciences

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Ribonucleic acid (RNA) hairpins are among the most common RNA secondary structural elements that are frequently capped by tetraloops. An RNA tetraloop structure is composed of a Watson–Crick base‐paired stem and four loop nucleotides. The structure is compact and stable. The first and the fourth nucleotides form a base pair in most of the tetraloops, leaving two unpaired nucleotides in the loop. A sharp turn in the backbone is stabilized by ribose–base, base–phosphate hydrogen bonds and base stacking interactions. Tetraloops help in the folding of RNA by initiating the process. Additionally, they provide sites for RNA tertiary contacts and for protein binding, facilitating the assembly of ribonucleoprotein particles. Certain interactions, some of which are sequence‐specific and others structure‐specific, can be involved in the recognition of tetraloops by proteins and RNAs. Key concepts: RNA hairpins play important structural and functional roles in RNA. Tetraloops help the folding of RNA and provide sites for RNA–RNA and RNA–protein interactions. U turn motif, a sharp backbone turn in tetraloops, is stabilized by ribose–base, base–phosphate hydrogen bonds and base stacking interactions.

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Linkage between Substrate Recognition and Catalysis during Cleavage of Sarcin/Ricin Loop RNA by Restrictocin

Cited by 18 publications

References 33 publications

Dissection of the high rate constant for the binding of a ribotoxin to the ribosome

Dissection of the high rate constant for the binding of a ribotoxin to the ribosome

Nondenaturing Purification of Co-Transcriptionally Folded RNA Avoids Common Folding Heterogeneity

RNAStructure: Tetraloops

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