Biomedical subjects

Kevin M Weeks

Publications and source records attributed to Kevin M Weeks.

25 records · Page 2Linked to original sources

PubMed2003

RNA-tethered phenyl azide photocrosslinking via a short-lived indiscriminant electrophile.

Arylazide mediated photocrosslinking has been widely used to obtain structural constraints in biological systems, even though the reactive species generated upon photolysis in aqueous solution have not been well characterized. We establish a mechanistic framework for formation of adducts between photoactivated 3-hydroxyphenyl azide and RNA. Tethered to an internal site in an RNA duplex via a 2'-amido linkage, photolysis of the aryl azide yields a cross-strand cross-link. Analysis of the ability of reagents with diagnostic reactivities to intercept formation of this cross-strand cross-link supports the assignment that the photoactivated intermediate is the ketenimine or a ketenimine-derived ring expansion product. Neither the initially produced singlet nitrene nor the subsequently formed triplet nitrene contribute to cross-link formation. Argon matrix and time-resolved solution experiments show that photolysis of free 3-hydroxyphenyl azide releases (in <or=20 ns) either a ketenimine or azepinone intermediate that reacts with nucleophiles. Adenosine, uridine, and guanosine monophosphate nucleotides have approximately equivalent abilities to quench the cross-strand cross-link, indicating that the photoactivated intermediate reacts broadly with functional groups in RNA. The reactive intermediate forms an adduct with adenosine monophosphate when tethered to both an RNA duplex or unstructured single strand; thus, cross-link formation is independent of the local RNA environment. The lifetime of the reactive intermediate generated upon photolysis of free 3-hydroxyphenyl azide in 50 mM Hepes diamine buffer is found to be 60-160 micros or significantly shorter than large scale RNA folding events. RNA-tethered 3-hydroxyphenyl azide cross-linking in aqueous buffer can thus be used with confidence to map structural neighbors, including most dynamic interactions, in RNA.

Acrylamide↗

PubMed2003

Kinetic and thermodynamic framework for assembly of the six-component bI3 group I intron ribonucleoprotein catalyst.

The yeast mitochondrial bI3 group I intron RNA splices in vitro as a six-component ribonucleoprotein complex with the bI3 maturase and Mrs1 proteins. We report a comprehensive framework for assembly of the catalytically active bI3 ribonucleoprotein. (1) In the absence of Mg(2+), two Mrs1 dimers bind independently to the bI3 RNA. The ratio of dissociation to association rate constants, k(off)/k(on), is approximately equal to the observed equilibrium K(1/2) of 0.12 nM. (2) At magnesium ion concentrations optimal for splicing (20 mM), two Mrs1 dimers bind with strong cooperativity to the bI3 RNA. k(off)/k(on) is 15-fold lower than the observed K(1/2) of 11 nM, which reflects formation of an obligate intermediate involving one Mrs1 dimer and the RNA in cooperative assembly of the Mrs1-RNA complex. (3) The bI3 maturase monomer binds to the bI3 RNA at almost the diffusion-controlled limit and dissociates with a half-life of 1 h. k(off)/k(on) is approximately equal to the equilibrium K(D) of 2.8 pM. The bI3 maturase thus represents a rare example of a group I intron protein cofactor whose binding is adequately characterized by a one-step mechanism under conditions that promote splicing. (4) Maturase and Mrs1 proteins each bind the bI3 RNA tightly, but with only modest coupling (approximately 1 kcal/mol), suggesting that the proteins interact at independent RNA binding sites. Maturase binding functions to slow dissociation of Mrs1; whereas prior Mrs1 binding increases the bI3 maturase k(on) right to the diffusion limit. (5) At effective concentrations plausibly present in yeast mitochondria, a predominant assembly pathway emerges involving rapid, tight binding by the bI3 maturase, followed by slower, cooperative assembly of two Mrs1 dimers. In the absence of other factors, disassembly of all protein subunits will occur in a single apparent step, governed by dissociation of the bI3 maturase.

Catalysis↗

PubMed2003

Small structural costs for evolution from RNA to RNP-based catalysis.

Typical RNA-based cellular catalysts achieve their active structures only as complexes with protein cofactors, implying that protein binding compensates for some structural deficiencies in the RNA. An unresolved question was the extent to which protein-facilitation imposes additional structural costs, by requiring that an RNA maintain structures required for protein binding, beyond those required for catalysis. We used nucleotide analog interference to identify initially 71 functional group substitutions at phosphate, 2'-ribose, and adenosine base positions that compromise RNA self-splicing in the bI5 group I intron. Protein-facilitated splicing by CBP2 suppresses 11 of 30 interfering substitutions at the RNA backbone and a greater fraction, 27 of 41, at the adenosine base, including at structures conserved among group I introns. Only one substitution directly interferes with protein binding but not with self-splicing. This substitution, plus three adenosine base modifications that interfere more strongly in CBP2-dependent splicing than in self-splicing, yield a cost for protein facilitation of only four functional groups, as approximated by this set of analogs. The small observed structural cost provides a strong physical rationale for the evolutionary drive from RNA to RNP-based function in biology. Remarkably, the four extra requirements do not appear to report disruption of direct protein-RNA contacts and instead likely reflect design against misfolding rather than for maintenance of a protein-binding site.

Animals↗

PubMed2003

Differential helix stabilities and sites pre-organized for tertiary interactions revealed by monitoring local nucleotide flexibility in the bI5 group I intron RNA.

The local environment at adenosine residues in the bI5 group I intron RNA was monitored as a function of Mg(2+) using both the traditional method of dimethyl sulfate (DMS) N1 methylation and a new approach, selective acylation of 2'-amine substituted nucleotides. These probes yield complementary structural information because N1 methylation reports accessibility at the base pairing face, whereas 2'-amine acylation scores overall residue flexibility. 2'-Amine acylation robustly detects RNA secondary structure and is sensitive to higher order interactions not monitored by DMS. Disruption of RNA structure due to the 2'-amine substitution is rare and can be compensated by stabilizing folding conditions. Peripheral helices that do not interact with other parts of the RNA are more stable than both base paired helices and tertiary interactions in the conserved catalytic core. The equilibrium state of the bI5 intron RNA, prior to assembly with its protein cofactor, thus features a relatively loosely packed core anchored by more stable external stem-loop structures. Adenosine residues in J4/5 and P9.0 form structures in which the nucleotide is constrained but the N1 position is accessible, consistent with pre-organization to form long-range interactions with the 5' and 3' splice sites.

Acylation↗

PubMed2002

Catalysis of amide synthesis by RNA phosphodiester and hydroxyl groups.

The functional groups found among the RNA bases and in the phosphoribose backbone represent a limited repertoire from which to construct a ribozyme active site. This work investigates the possibility that simple RNA phosphodiester and hydroxyl functional groups could catalyze amide bond synthesis. Reaction of amine groups with activated esters would be catalyzed by a group that stabilizes the partial positive charge on the amine nucleophile in the transition state. 2'-Amine substitutions adjacent to 3'-phosphodiester or 3'-hydroxyl groups react efficiently with activated esters to form 2'-amide and peptide products. In contrast, analogs in which the 3'-phosphodiester is replaced by an uncharged phosphotriester or is constrained in a distal conformation react at least 100-fold more slowly. Similarly, a nucleoside in which the 3'-hydroxyl group is constrained trans to the 2'-amine is also unreactive. Catalysis of synthetic reactions by RNA phosphodiester and ribose hydroxyl groups is likely to be even greater in the context of a preorganized and solvent-excluding catalytic center. One such group is the 2'-hydroxyl of the ribosome-bound P-site adenosine substrate, which is close to the amine nucleophile in the peptidyl synthesis reaction. Given ubiquitous 2'-OH groups in RNA, there exists a decisive advantage for RNA over DNA in catalyzing reactions of biological significance.

Acylation↗

PubMed2002

Chemical interrogation of mismatches in DNA-DNA and DNA-RNA duplexes under nonstringent conditions by selective 2'-amine acylation.

2'-Amine-substituted nucleotides in hybridized duplexes can be chemically tagged in an acylation reaction that is faster for mismatched or flexible nucleotides than for residues constrained by base pairing. Here we explore mismatch and hybridization detection using probe oligodeoxynucleotides containing single 2'-aminocytidine or -uridine nucleotides annealed to DNA or RNA targets under nonstringent conditions, below T(m). Consistent with a mechanism in which 2'-amine acylation is gated by local nucleotide flexibility, we find that efficient acylation is correlated with formation of weaker or fewer hydrogen bonds in base pair mismatches. Using 2'-aminocytidine-containing probes annealed to both DNA and RNA targets, mismatches are reliably detected as rapid selective acylation of the 2'-amine group in two sequence contexts. For probe oligonucleotides containing 2'-aminouridine residues, good discrimination between U-A base pairs and U-G mismatches could be obtained for DNA-DNA but not for DNA-RNA duplexes upon the introduction of a single 2'-O-Me group 5' to the 2'-amino nucleotide. The 2'-O-Me group introduces a structural perturbation, presumably to a more A-form-like structure, that exaggerates local flexibility at mismatches in DNA strands. Thus, 2'-amine acylation can be used to interrogate all possible mismatches in DNA-DNA duplexes and mismatches involving 2'-amine-substituted cytidine nucleotides in DNA-RNA heteroduplexes. Applications of this chemistry include detecting and chemically proofreading single nucleotide polymorphisms in both DNA and RNA targets and quantifying absolute amounts of RNA.

Acylation↗

PubMed2002

Recruitment of intron-encoded and co-opted proteins in splicing of the bI3 group I intron RNA.

Detectable splicing by the Saccharomyces cerevisiae mitochondrial bI3 group I intron RNA in vitro is shown to require both an intron-encoded protein, the bI3 maturase, and the nuclear-encoded protein, Mrs1. Both proteins bind independently to the bI3 RNA. The bI3 maturase binds as a monomer, whereas Mrs1 is a dimer in solution that assembles as two dimers, cooperatively, on the RNA. The active six-subunit complex has a molecular mass of 420 kDa, splices with a k(cat) of 0.3 min(-1), and binds the guanosine nucleophile with an affinity comparable to other group I introns. The functional bI3 maturase domain is translated from within the RNA that encodes the intron, has evolved a high-affinity RNA-binding activity, and is a member of the LAGLIDADG family of DNA endonucleases, but appears to have lost DNA cleavage activity. Mrs1 is a divergent member of the RNase H fold superfamily of dimeric DNA junction-resolving enzymes that also appears to have lost its nuclease activity and now functions as a tetramer in RNA binding. Thus, the bI3 ribonucleoprotein is the product of a process in which a once-catalytically active RNA now obligatorily requires two facilitating protein cofactors, both of which are compromised in their original functions.

Amino Acid Sequence↗