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Y Shamoo

Publications and source records attributed to Y Shamoo.

17 recordsLinked to original sources
PubMed2006

Protein-RNA interactions: exploring binding patterns with a three-dimensional superposition analysis of high resolution structures.

MOTIVATION: The recognition of specific RNA sequences and structures by proteins is critical to our understanding of RNA processing, gene expression and viral replication. The diversity of RNA structures suggests that RNA recognition is substantially different than that of DNA. RESULTS: The atomic coordinates of 41 protein-RNA complexes have been used to probe composite nucleoside binding pockets that form the structural and chemical underpinnings of base recognition. Composite nucleoside binding pockets were constructed using three-dimensional superpositions of each RNA nucleoside. Unlike protein-DNA interactions which are dominated by accessibility, RNA recognition frequently occurs in non-canonical and single-strand-like structures that allow interactions to occur from a much wider set of geometries and make fuller use of unique base shapes and hydrogen-bonding ability. By constructing composites that include all van der Waals, hydrogen-bonding, stacking and general non-polar interactions made to a particular nucleoside, the strategies employed are made readily visible. Protein-RNA interactions can result in the formation of a glove-like tight binding pocket around RNA bases, but the size, shape and non-polar binding patterns differ between specific RNA bases. We show that adenine can be distinguished from guanine based on the size and shape of the binding pocket and steric exclusion of the guanine N2 exocyclic amino group. The unique shape and hydrogen-bonding pattern for each RNA base allow proteins to make specific interactions through a very small number of contacts, as few as two in some cases. AVAILABILITY: The program ENTANGLE is available from http://www.bioc.rice.edu/~shamoo

Algorithms↗
PubMed2001

Structure-based analysis of protein-RNA interactions using the program ENTANGLE.

Until recently, drawing general conclusions about RNA recognition by proteins has been hindered by the paucity of high-resolution structures. We have analyzed 45 PDB entries of protein-RNA complexes to explore the underlying chemical principles governing both specific and non-sequence specific binding. To facilitate the analysis, we have constructed a database of interactions using ENTANGLE, a JAVA-based program that uses available structural models in their PDB format and searches for appropriate hydrogen bonding, stacking, electrostatic, hydrophobic and van der Waals interactions. The resulting database of interactions reveals correlations that suggest the basis for the discrimination of RNA from DNA and for base-specific recognition. The data illustrate both major and minor interaction strategies employed by families of proteins such as tRNA synthetases, ribosomal proteins, or RNA recognition motifs with their RNA targets. Perhaps most surprisingly, specific RNA recognition appears to be mediated largely by interactions of amide and carbonyl groups in the protein backbone with the edge of the RNA base. In cases where a base accepts a proton, the dominant amino acid donor is arginine, whereas in cases where the base donates a proton, the predominant acceptor is the backbone carbonyl group, not a side-chain group. This is in marked contrast to DNA-protein interactions, which are governed predominantly by amino acid side-chain interactions with functional groups that are presented in the accessible major groove. RNA recognition often proceeds through loops, bulges, kinks and other irregular structures that permit use of all the RNA functional groups and this is seen throughout the protein-RNA interaction database.

Adenine↗
PubMed1999

Building a replisome from interacting pieces: sliding clamp complexed to a peptide from DNA polymerase and a polymerase editing complex.

We have solved the crystal structures of the bacteriophage RB69 sliding clamp, its complex with a peptide essential for DNA polymerase interactions, and the DNA polymerase complexed with primer-template DNA. The editing complex structure shows a partially melted duplex DNA exiting from the exonuclease domain at an unexpected angle and significant changes in the protein structure. The clamp complex shows the C-terminal 11 residues of polymerase bound in a hydrophobic pocket, and it allows docking of the editing and clamp structures together. The peptide binds to the sliding clamp at a position identical to that of a replication inhibitor peptide bound to PCNA, suggesting that the replication inhibitor protein p21CIP1 functions by competing with eukaryotic polymerases for the same binding pocket on the clamp.

Amino Acid Sequence↗
PubMed1997

Crystal structure of the two RNA binding domains of human hnRNP A1 at 1.75 A resolution.

Heterogeneous ribonucleoprotein A1 (hnRNP A1) is an abundant eukaryotic nuclear RNA binding protein. A1 is involved in the packaging of pre-mRNA into hnRNP particles, transport of poly A+ mRNA from the nucleus to the cytoplasm and may modulate splice site selection. The crystal structure of A1(RBD1,2) reveals two independently-folded RNA binding domains (RBDs) connected by a flexible linker. Both RBDs are structurally homologous to the U1A(RBD1), and have their RNA binding platforms oriented in an anti-parallel fashion. The anti-parallel arrangement of the A1 RNA binding platforms suggests mechanisms for RNA condensation and ways of bringing together distant RNA sequences for RNA metabolism such as splicing or transport.

Binding Sites↗
PubMed1995

Crystal structure of a replication fork single-stranded DNA binding protein (T4 gp32) complexed to DNA.

The single-stranded DNA (ssDNA) binding protein gp32 from bacteriophage T4 is essential for T4 DNA replication, recombination and repair. In vivo gp32 binds ssDNA as the replication fork advances and stimulates replisome processivity and accuracy by a factor of several hundred. Gp32 binding affects nearly every major aspect of DNA metabolism. Among its important functions are: (1) configuring ssDNA templates for efficient use by the replisome including DNA polymerase; (2) melting out adventitious secondary structures; (3) protecting exposed ssDNA from nucleases; and (4) facilitating homologous recombination by binding ssDNA during strand displacement. We have determined the crystal structure of the gp32 DNA binding domain complexed to ssDNA at 2.2 A resolution. The ssDNA binding cleft comprises regions from three structural subdomains and includes a positively charged surface that runs parallel to a series of hydrophobic pockets formed by clusters of aromatic side chains. Although only weak electron density is seen for the ssDNA, it indicates that the phosphate backbone contacts an electropositive cleft of the protein, placing the bases in contact with the hydrophobic pockets. The DNA mobility implied by the weak electron density may reflect the role of gp32 as a sequence-independent ssDNA chaperone allowing the largely unstructured ssDNA to slide freely through the cleft.

Amino Acid Sequence↗
PubMed1995

Multiple RNA binding domains (RBDs) just don't add up.

One of the most common motifs for binding RNA in eukaryotes is the RNA binding domain (RBD) or RNA Recognition Motif (RRM). One of the more intriguing aspects of these proteins is their modular nature. Proteins have been found containing from one to four RRMs. In most instances, these domains have some basal level of non-sequence specific RNA binding affinity. In addition, many also have a higher affinity for a specific structure or sequence of RNA. In the cases of heterogenous nuclear ribonucleoprotein A1 (hnRNP A1), yeast poly-A binding protein and splicing factor U2AF65, the individual free energy of binding of the RBDs for RNA are not strictly additive. By invoking a model in which the amino acids connecting adjoining RBDs are considered to be flexible linkers with an interresidue spacing of about 3.5 A, it is possible to predict the apparent association constants for at least some multi-RBD proteins to single-stranded RNA. We have surveyed the literature and found that individual RBDs are separated by 'linker' sequences of highly variable length. These linkers provide a critical determinant of binding affinity and may modulate cis versus trans binding. A clearer understanding of multi-RBD binding is essential to critically evaluating the role of these proteins in RNA splicing, packaging and transport.

Amino Acid Sequence↗
PubMed1994

Both RNA-binding domains in heterogenous nuclear ribonucleoprotein A1 contribute toward single-stranded-RNA binding.

Heterogenous nuclear ribonucleoproteins (hnRNPs) such as hnRNP A1 are tightly associated with heterogenous nuclear RNAs (hnRNAs) within eukaryotic nuclei and are thought to be involved in hnRNA processing and splice site selection. The NH2-terminal two-thirds of hnRNP A1 contains two 92-amino acid RNA binding domains (RBDs) that are arranged in tandem and are more than 30% homologous with each other. Following this region is a flexible glycine-rich COOH-terminal domain. We have studied the nucleic acid binding properties of the two isolated RBDs (residues 1-92 and 93-184, respectively) and of A1 fragments corresponding to residues 1-184 and 1-196 (i.e., the latter fragment is called UP1) in order to evaluate their relative contributions to A1 binding. We have determined that the individual RBDs of A1 bind poly[r(epsilon A)], a fluorescent single-stranded RNA (ssRNA), with a surprisingly low apparent association constant of only 1.5 x 10(4) M-1 (1-92) and 4.5 x 10(4) M-1 (93-184), respectively. We hypothesize that this low affinity represents a basal level of binding that is common to most RBD-containing proteins. Oligonucleotide binding studies suggest the interaction site size for the 93-184 fragment is approximately 4 nucleotides or less and salt sensitivity studies indicate that only about 27% of the free energy of binding of this RBD derives from ionic interactions. Since the affinity of the 1-184 fragment is at least 10-fold above that of either of its component RBDs, both must contribute to binding. This conclusion is further supported by the increased occluded site size of 1-184 (n = 14 +/- 2), as compared to its 93-184 RBD (n = 6 +/- 1), and by the biphasic binding that was observed for the UP1:poly(U) interaction at pH 6.0. Our finding that the affinity of the 1-184 fragment is 1000-fold less than the product of the affinities of its 1-92 and 93-184 RBDs is consistent with these domains being joined by a flexible linker. By comparing the affinities of the 1-184 fragment with that for A1, we conclude that together the two RBDs in A1 account for only 53% of the free energy of A1 binding. Comparative binding studies with UP1 demonstrate that the short region spanning residues 185-->195 represents an important determinant of the binding affinity of A1 and, since this region contains a site of dimethylation, it may provide a mechanism for regulating the affinity of A1 for specific nucleic acid targets.

Amino Acid Sequence↗
PubMed1994

Determination of the secondary structure and folding topology of an RNA binding domain of mammalian hnRNP A1 protein using three-dimensional heteronuclear magnetic resonance spectroscopy.

The secondary structure and folding topology of the first RNA binding domain of the human hnRNP A1 protein was determined by multidimensional heteronuclear NMR spectroscopy. The 92 amino acid long domain exhibits a beta alpha beta beta alpha beta folding pattern, arranged in a four-stranded antiparallel beta-sheet flanked by two alpha-helices, which is very similar to that found for other members of this family. Regions of marked variation between the structurally characterized RNA binding proteins of this class to date are mainly localized in the loops connecting the secondary structure elements.

Amino Acid Sequence↗
PubMed1993

Translational repression by the bacteriophage T4 gene 32 protein involves specific recognition of an RNA pseudoknot structure.

An RNA pseudoknot has been shown to form the 5'-end of bacteriophage T4 gene 32 mRNA that is essential to autoregulation of gene 32 (g32) mRNA by gene product 32 (gp32), a single-stranded nucleic acid binding protein. Structure-mapping of RNA oligonucleotides with structure-specific RNases indicate that two stem regions consisting of nucleotides -67 to -64 base-paired to -52 to -55 (stem-1) and nucleotides -62 to -56 base-paired to -40 to -46 (stem-2) can fold into a "pseudoknotted" structure that may be analogous to the semi-continuous a-helical pseudoknot. Our results suggest that the g32 mRNA pseudoknot can form under conditions where specific autoregulation by gp32 is observed. Although the g32 mRNA pseudoknot is stabilized by Mg2+, it exists in equilibrium with a 3'-hairpin structure. Gel mobility studies carried out with defined length oligonucleotides indicate the gp32 does, in fact, bind tightly to the pseudoknot. These studies agree with the proposal of McPheeters et al., that the pseudoknot represents a nucleation site essential for autogenous gp32 translation regulation. Although disruption of tertiary structure interactions in this pseudoknot (with EDTA) significantly reduces the ability of gp32 to specifically recognize its own mRNA, in vitro mutagenesis studies suggest the sequence of stem-2 and of the loop region (nucleotides -47 to -50) also represent important determinants for specific gp32 autoregulation. Based on a competition assay relying on gel mobility shifts, the order of importance of the major elements of the pseudoknot are stem-1 > sequence of stem-2 or loop-2 > stem-2. In this assay, disruption of stem-1 decreased the ability of the resulting structure to compete for gp32 binding by approximately fourfold. Both stem-1 and stem-2 appear to be essential to maintain high-level expression from gp32 mRNA in an in vitro transcription/translation system. Taken together, these results support the translation control model in which the pseudoknot region is a nucleation point for cooperative gp32 binding, which then proceeds in a 3' direction through a long stretch of single-stranded RNA that includes the initiation codon for gene 32.

Bacteriophage T4↗
PubMed1991

A retrovirus-like zinc domain is essential for translational repression of bacteriophage T4 gene 32.

Gene 32 protein (gp32), a single-stranded DNA-binding protein from bacteriophage T4, contains a zinc-binding subdomain with sequence homologies to the 3-cysteine/1-histidine zinc-binding motif found in a variety of retroviruses and plant viruses. In vitro studies suggest that autoregulation of gp32 occurs at the level of translation by gp32 specifically binding gene 32 mRNA at an unusual stem-loop structure that can be modeled as an RNA pseudoknot. Nucleation of gp32 binding via this pseudoknot is thought to be needed to facilitate cooperative binding of gp32 through a largely unstructured region that overlaps the ribosome binding site (McPheeters, D. S., Stormo, G. D., and Gold, L. (1988) J. Mol. Biol. 201, 517-535). Removal of Zn(II) from gp32 results in a protein that retains the ability to bind single-stranded RNA with high affinity but is unable to specifically autoregulate itself at the level of translation. Deletion of the pseudoknot sequences from the gene 32 autoregulatory region results in an mRNA that cannot be repressed by gp32. These results suggest that the zinc-binding subdomain of gp32 plays an essential role in autoregulation by providing a critical element necessary for nucleating cooperative binding at the gene 32 mRNA pseudoknot.

Amino Acid Sequence↗
PubMed1991

A rapid method for purification of synthetic oligoribonucleotides.

A procedure for large-scale purification of synthetic oligoribonucleotides has been developed that has significant advantages over gel purification techniques currently in use. Synthesis was performed using commercially available 2'-O-silylated ribonucleoside 3'-O-phosphoramidites, and coupling efficiencies were consistently greater than 97% for oligoribonucleotides up to 31 residues in length. Using C4 reverse-phase chromatography to remove material not deprotected by treatment with tetrabutylammonium fluoride, we have eliminated reactants in which the 2'-O-silyl group is only partly removed, thus ensuring a homogeneous population of oligoribonucleotide.

Chromatography, High Pressure Liquid↗
PubMed1990

A novel function for zinc(II) in a nucleic acid-binding protein. Contribution of zinc(II) toward the cooperativity of bacteriophage T4 gene 32 protein binding.

The contribution of Zn(II) toward the binding of bacteriophage T4 gene 32 single-stranded nucleic acid-binding protein (gp32) has been examined by the use of two independent approaches. Studies carried out with successively longer oligonucleotides which have the general structure p(dT)n, where n is equal to 8, 16, 24, or 32 nucleotides, suggest that removal of Zn(II) decreases the cooperativity of binding by as much as 30-fold. Hence, whereas apo-gp32 and native gp32 have similar apparent affinities for the single-site lattice p(dT)8, native gp32 has an approximately 10-fold higher affinity compared to apo-gp32 for a two-site lattice, such as p(dT)16. In contrast to native gp32, where full cooperativity (in terms of the strength of a single gp32-gp32 interaction) is reached with only a two-site lattice, the cooperativity of apo-gp32 binding appears to increase approximately 4-fold upon going from a two- to a four-site lattice such as p(dT)32. The conclusion reached from these oligonucleotide studies agrees well with a series of titrations with polyribo(ethenoadenylic) acid, in 0.275-0.40 M NaCl. These latter studies indicate that the 6-38-fold higher affinity of native gp32 as compared to apo-gp32 for polyribo(ethenoadenylic) acid results primarily from the higher cooperativity of binding of native gp32. By stabilizing a specific subdomain within gp32 that is essential along with the NH2-terminal domain (residues 1-9), Zn(II) contributes from 20 to 50% of the free energy of cooperative gp32-gp32 interactions that occur along a polynucleotide lattice.

Amino Acid Sequence↗
PubMed1989

Site-specific mutagenesis of T4 gene 32: the role of tyrosine residues in protein-nucleic acid interactions.

Bacteriophage T4 gene 32 encodes a single-stranded DNA (ssDNA) binding protein (gp32) required for T4 DNA replication, recombination, and repair. Previous physicochemical studies on gp32 and other ssDNA binding proteins have suggested that binding may involve hydrophobic interactions that result from the close approach of several aromatic amino acid side chains with the nucleic acid bases. In the case of gp32, five tyrosines and two phenylalanines have previously been implicated in gp32.ssDNA complex formation. Site-directed mutagenesis of T4 gene 32 was employed to produce a set of eight gp32 mutant proteins, each of which encoded a single substitution at one of the eight tyrosine residues within gp32. The mutant gp32 proteins were then subjected to physicochemical analysis to evaluate the role of each tyrosine residue in gp32 structure and function. Oligonucleotide binding studies suggest that tyrosine residues 84, 99, 106, 115, and 186 each contribute from 0.3 to 0.7 kcal/mol to ssDNA binding, which corresponds to 3-7% of the overall binding energy for gp32.ssDNA complex formation. Replacement of tyrosine residues 73 and 92 appears to lead to large structural changes that may be the result of disrupting the zinc binding subdomain within gp32.

Calorimetry, Differential Scanning↗
PubMed1988

Photochemical crosslinking of bacteriophage T4 single-stranded DNA-binding protein (gp32) to oligo-p(dT)8: identification of phenylalanine-183 as the site of crosslinking.

Using ultraviolet light, both the 33,000-dalton single-stranded DNA-binding protein from T4 bacteriophage (gp32) as well as a 25,000-dalton limited trypsin cleavage product of gp32 (core gp32*) that retains high affinity for single-stranded DNA can be crosslinked to an oligodeoxynucleotide, p(dT)8. After photolysis, a single tryptic peptide crosslinked to p(dT)8 was isolated by anion-exchange high-performance liquid chromatography. Gas-phase sequencing of this modified peptide gave the following sequence: Gln-Val-Ser-Gly-(X)-Ser-Asn-Tyr-Asp-Glu-Ser-Lys, which corresponds to residues 179-190 in gp32. Based on the absence of the expected phenylthiohydantoin derivative of phenylalanine 183 at cycle 5 (X) we infer that crosslinking has occurred at this position and that phenylalanine 183 is at the interface of the gp32:p(dT)8 complex in an orientation that allows covalent bond formation with the thymine radical produced by ultraviolet irradiation.

Cross-Linking Reagents↗
PubMed1986

1H NMR (500 MHz) identification of aromatic residues of gene 32 protein involved in DNA binding by use of protein containing perdeuterated aromatic residues and by site-directed mutagenesis.

Preparation of gene 32 protein containing perdeuterated tyrosyl and phenylalanyl residues has allowed the resolution of separate 1H NMR signals for the Tyr and Phe residues of the protein by NMR difference spectra. Upfield shifts in the chemical shifts of a number of aromatic protons previously observed to accompany deoxyoligonucleotide complex formation with gene 32 protein [Prigodich, R. V., Casas-Finet, J., Williams, K. R., Konigsberg, W., & Coleman, J. E. (1984) Biochemistry 23, 522-529] can be assigned to five Tyr and two Phe residues that must form part of the DNA binding domain. Site-directed mutation of Tyr-115 to Ser-115 results in the disappearance of a set of 2,6 and 3,5 tyrosyl protons that are among those moved upfield by oligonucleotide complex formation. These findings suggest that the amino acid sequence from Tyr-73 to Tyr-115 which contains six of the eight Tyr residues of the protein forms part of the DNA binding surface.

Amino Acid Sequence↗
PubMed1986

Cloning of T4 gene 32 and expression of the wild-type protein under lambda promoter PL regulation in Escherichia coli.

Bacteriophage T4 gene 32 encodes a single-stranded DNA binding protein required for T4 DNA replication, recombination, and repair. Previous attempts at cloning gene 32 have failed due to a presumably deleterious effect on host cell viability. In addition, overexpression of gene 32 would be expected to be limited by the autoregulatory ability of the gene 32 product g32P. A repetitive A + T-rich sequence flanking the ribosome binding site of gene 32 has been implicated in this translational regulation. To circumvent these problems, the wild-type gene for g32P has been reconstructed in M13 using restriction fragments from T4 g32am453 and synthetic oligodeoxynucleotides so that it no longer includes its native promoter and putative autoregulatory region. The g32am453 codon TAG was changed back to TGG as in wild-type gene 32 using site-directed oligodeoxynucleotide mutagenesis. In vectors containing the lambda leftward promoter PL, gene 32 is overexpressed with the resulting transcripts being depressed at g32P concentrations that repress the wild-type gene 32 transcripts.

Bacteriophage lambda↗