Biomedical subjects

C Zwieb

Publications and source records attributed to C Zwieb.

At least 19 recordsLinked to original sources

PubMed1999

Crystal structure of the conserved subdomain of human protein SRP54M at 2.1 A resolution: evidence for the mechanism of signal peptide binding.

Protein SRP54 is an integral part of the mammalian signal recognition particle (SRP), a cytosolic ribonucleoprotein complex which associates with ribosomes and serves to recognize, bind, and transport proteins destined for the membrane or secretion. The methionine-rich M-domain of protein SRP54 (SRP54M) binds the SRP RNA and the signal peptide as the nascent protein emerges from the ribosome. A focal point of this critical cellular function is the detailed understanding of how different hydrophobic signal peptides are recognized efficiently and transported specifically, despite considerable variation in sequence. We have solved the crystal structure of a conserved functional subdomain of the human SRP54 protein (hSRP54m) at 2.1 A resolution showing a predominantly alpha helical protein with a large fraction of the structure available for binding. RNA binding is predicted to occur in the vicinity of helices 4 to 6. The N-terminal helix extends significantly from the core of the structure into a large but constricted hydrophobic groove of an adjacent molecule, thus revealing molecular details of possible interactions between alpha helical signal peptides and human SRP54.

Amino Acid Sequence↗

PubMed1999

Comparative sequence analysis of tmRNA.

Minimal secondary structures of the bacterial and plastid tmRNAs were derived by comparative analyses of 50 aligned tmRNA sequences. The structures include 12 helices and four pseudoknots and are refinements of earlier versions, but include only those base pairs for which there is comparative evidence. Described are the conserved and variable features of the tmRNAs from a wide phylogenetic spectrum, the structural properties specific to the bacterial subgroups and preliminary 3-dimensional models from the pseudoknotted regions.

Bacteria↗

PubMed1999

The tmRNA database (tmRDB).

As of September, 1998, a total of 43 sequences are contained within the tmRNA database (tmRDB). The tmRNA sequences are arranged alphabetically and ordered phylogenetically. The alignment of the tmRNAs emphasizes the basepairs that are supported by comparative sequence analysis and establishes minimal secondary structures for the known tmRNAs. A corresponding alignment of the predicted tmRNA-encoded tag peptides is presented. The tmRDB also offers a small number of RNA secondary structure diagrams and PDB-formatted three-dimensional models generated with the program ERNA-3D. The data are available freely at the URL http://psyche.uthct.edu/dbs/tmRDB/tmRDB.++ +html

Bacteria↗

PubMed1999

The Signal Recognition Particle Database (SRPDB).

The signal recognition particle database (SRPDB) is located at the University of Texas Health Science Center at Tyler and includes tabulations of SRP RNA, SRP protein and SRP receptor sequences. The sequences are annotated with links to the primary databases. They are ordered alphabetically or phylogenetically and are available in aligned form. As of September, 1998, there were 108 SRP RNA sequences, 83 SRP protein sequences and 28 sequences of the SRP receptor alpha subunit and its homologues. In addition, the SRPDB provides search motifs consisting of conserved amino acid and nucleotide residues, and a limited number of SRP RNA secondary structure diagrams and 3-D models. The data are available freely at the URL http://psyche.uthct.edu/dbs/SRPDB/SRPDB.++ +html

Academic Medical Centers↗

PubMed1999

Expression, purification, and crystallography of the conserved methionine-rich domain of human signal recognition particle 54 kDa protein.

Protein SRP54 is an essential component of eukaryotic signal recognition particle (SRP). The methionine-rich M-domain (SRP54M or 54M) interacts with the SRP RNA and is also involved in the binding to signal peptides of secretory proteins during their targeting to cellular membranes. To gain insight into the molecular details of SRP-mediated protein targeting, we studied the human 54M polypeptide. The recombinant human protein was expressed successfully in Escherichia coli and was purified to homogeneity. Our studies determined the sites that were susceptible to limited proteolysis, with the goal to design smaller functional mutant derivatives that lacked nonessential amino acid residues from both termini. Of the four polypeptides produced by V8 protease or chymotrypsin, 54MM-2 was the shortest (120 residues; Mr = 13,584.8), but still contained the conserved amino acids suggested to associate with the signal peptide or the SRP RNA. 54MM-2 was cloned, expressed, purified to homogeneity, and was shown to bind human SRP RNA in the presence of protein SRP19, indicating that it was functional. Highly reproducible conditions for the crystallization of 54MM-2 were established. Examination of the crystals by X-ray diffraction showed an orthorhombic unit cell of dimensions a = 29.127 A, b = 63.693 A, and c = 129.601 A, in space group P2(1)2(1)2(1), with reflections extending to at least 2.0 A.

Amino Acid Sequence↗

PubMed1998

Protein SRP54 of human signal recognition particle: cloning, expression, and comparative analysis of functional sites.

Signal recognition particle (SRP) plays a critical role in the targeting of secretory proteins to cellular membranes. An essential component of SRP is the protein SRP54, which interacts not only with the nascent signal peptide, but also with the SRP RNA. To understand better how protein targeting occurs in the human system, the human SRP54 gene was cloned, sequenced, and the protein was expressed in bacteria and insect cells. Recombinant SRP54 was purified from both sources. The protein bound to SRP RNA in the presence of protein SRP19, and associated with the signal peptide of in vitro translated pre-prolactin. Comparative sequence analysis of human SRP54 with homologs from all three phylogenetic domains was combined with high-stringency protein secondary structure prediction. A conserved RNA-binding loop was predicted in the largely helical M-domain of SRP54. Contrary to general belief, the unusually high number of methionine residues clustered outside the predicted helices, thus indicating a mechanism of signal peptide recognition that may involve methionine-rich loops.

Amino Acid Sequence↗

PubMed1998

The tmRNA database (tmRDB).

This first release of the tmRNA database (tmRDB) contains 19 tmRNA sequences, a tmRNA sequence alignment with emphasis of base pairs that are supported by comparative sequence analysis, and a tabulation of tmRNA-encoded tag peptides. The tmRNADB also offers an RNA secondary structure diagram of the Escherichia coli tmRNA, as well as PDB-formatted coordinates for three-dimensional modeling. The data are available on the World Wide Web at http://www.uthct. edu/tmRDB/tmRDB.html

Computer Communication Networks↗

PubMed1998

The Signal Recognition Particle Database (SRPDB).

This release of the SRPDB (signal recognition particle database, http://pegasus.uthct.edu/SRPDB/SRPDB . html ) adds four SRP RNA sequences (a total of 99 SRP RNA sequences), 23 SRP protein sequences (a total of 63 protein sequences from SRP9, SRP14, SRP19, SRP21, SRP54, SRP68 or SRP72), and, for the first time, sequences of the alpha subunit of the eukaryotic SRP receptor and its homologous bacterial proteins (a total of 21 sequences). Sequences are offered phylogenetically ordered, annotated with links to the primary databases, and in aligned form. Also downloadable are sample SRP RNA secondary structure diagrams, three-dimensional models of representative SRP RNAs, and search motifs.

Amino Acid Sequence↗

PubMed1997

Determinants of a protein-induced RNA switch in the large domain of signal recognition particle identified by systematic-site directed mutagenesis.

Signal recognition particle (SRP) is a ribonucleoprotein complex that associates with ribosomes to promote the co-translational translocation of proteins across biological membranes. Human SRP RNA molecules exist in two distinct conformations, SR-A and SR-B, which may exchange during the assembly of the particle or could play a functional role in the SRP cycle. We have used systematic site-directed mutagenesis of the SRP RNA to determine the electrophoretic mobilities of altered RNA molecules, and we have identified the nucleotides that avert the formation of the two conformers. The conformer behavior of the various RNAs was addressed quantitatively by calculating a value zeta as an indicator of conformational variability. Single loose A-like forms were induced by changes in helix 5 [nucleotides (nt) at positions 111-128 or 222-231], helix 6 (nt at positions 141-150) and helix 7 (nt at position 169 and 170), whereas other mutations in helix 6 and helix 8 preserved the conformational variability of the mutant RNA molecules. The more compact B-like form was induced only when a small region (129-CAAUAU-134), located in the 5'-proximal portion of helix 6, was altered. Since this region is part of the binding site for SRP19, we suggest that protein SRP19 uses nucleotides at 129-134 to trigger the formation of the favored SR-B-form, and thus directs an early step in the hierarchical assembly of the large SRP domain.

Binding Sites↗

PubMed1997

Identification of an RNA-binding-loop in the N-terminal region of signal-recognition-particle protein SRP19.

Protein SRP19 is a 144-amino-acid polypeptide that associates intimately with the signal-recognition particle RNA (SRP RNA) and serves as an important structural and functional component of the SRP. We investigated the structure and RNA-binding activity of the human SRP19 protein by the use of comparative sequence analysis, high-stringency structure prediction, proteolytic susceptibility, and site-directed mutagenesis. SRP19 was found to consist of two distinct regions (called N-terminal and C-terminal regions) that are separated by a boundary of approximately 12-15 amino acid residues. Both regions contain an alpha-helix and several beta-strands that are connected by loops or turns. In agreement with the hypothetical model, proteolytic susceptibility demonstrated the predominant accessibility of two sites: one in a surface loop of the N-terminal region (YLNNKKTIAEGR33), and another site in the C-terminal tail at residues L129 and E133. The RNA-binding activities of mutant polypeptides with changes of conserved lysines and arginines (mutants K27Q, R33Q and R34Q) demonstrated that the proteolytically accessible loop of the N-terminal region is in direct contact with the SRP RNA. In contrast, alteration of a certain basic amino acid residues in the C-terminal region (R83, K116 and R118), as well as a deletion of four amino acid residues located at the boundary between the two regions, had no effect on the RNA-binding ability. The structural model that emerges from our data is thematically similar to that of ribosomal protein S5, the N-domain of which contains a loop motif believed to interact with double-stranded RNA. The presence of a similar structural feature in protein SRP19 has significant implications for the structure and function of the SRP19-RNA complex.

Amino Acid Sequence↗

PubMed1997

Binding site of the M-domain of human protein SRP54 determined by systematic site-directed mutagenesis of signal recognition particle RNA.

The interaction of protein SRP54M from the human signal recognition particle with SRP RNA was studied by systematic site-directed mutagenesis of the RNA molecule. Protein binding sites were identified by the analysis of mutations that removed individual SRP RNA helices or disrupted helical sections in the large SRP domain. The strongest effects on the binding activity of a purified polypeptide that corresponds to the methionine-rich domain of SRP54 (SRP54M) were caused by changes in helix 8 of the SRP RNA. Binding of protein SRP19 was diminished significantly by mutations in helix 6 and was stringently required for SRP54M to associate. Unexpectedly, mutant RNA molecules that resembled bacterial SRP RNAs were incapable of interaction with SRP54M, showing that protein SRP19 has an essential and direct role in the formation of the ternary complex with SRP54 and SRP RNA. Our findings provide an example for how, in eukaryotes, an RNA function has become protein dependent.

Base Sequence↗

PubMed1997

The uRNA database.

The uRNADB offers aligned, annotated and phylogenetically ordered sequences of several U RNAs. New to this release are RNAs from U7 (14 sequences), U8 (two sequences), U11 (three sequences), U12 (two sequences), U14 (11 sequences), U18, U48 and U49. A total of 34 new sequences were aligned with the previously compiled snRNAs U1, U2, U3, U4, U5 and U6.

Animals↗

PubMed1997

The Signal Recognition Particle Database (SRPDB).

The SRPDB (Signal Recognition Particle Database) offers aligned SRP RNA and SRP protein sequences, phylogenetically ordered and annotated. This release adds three SRP RNA sequences (totaling 96 SRP RNA sequences) and 11 SRP protein sequences (a total of 39 protein sequences from SRP9, SRP14, SRP19, SRP21, SRP54, SRP68 or SRP72). Also downloadable are sample SRP RNA secondary structure diagrams, a three-dimensional model of the human SRP RNA, search motifs and software.

Amino Acid Sequence↗

PubMed1997

Purification and biochemical characterization of the 19-kDa signal recognition particle RNA-binding protein expressed as a hexahistidine-tagged polypeptide in Escherichia coli.

The signal recognition particle (SRP) is a ribonucleoprotein complex that mediates translocation of proteins into the endoplasmic reticulum. Protein SRP19 is an essential structural component of SRP and is believed to promote assembly of the particle. In order to have a convenient source for the purification of milligram amounts of SRP19, we expressed in Escherichia coli a human SRP19 cDNA with an amino-terminal addition of six histidine residues. Expression at 25 degrees C eliminated formation of insoluble SRP19 and resulted in accumulation of soluble hexahistidine-SRP19 to 68% of total cell protein after 24 h. Metal chelation chromatography yielded 40 mg of hexahistidine-SRP19 per liter of culture, with a purity slightly greater than 97%. To examine protein function, the RNA-binding properties of the purified protein were determined by RNA electromobility shift assays. The histidine-tagged SRP19 bound specifically to a 150-nucleotide RNA derived from SRP RNA, with an apparent Kd of 1 nM, and bound, with greatly reduced affinity, to a mutagenized form of the SRP RNA derivative that contained an altered helix 6 tetranucleotide loop. The purified protein was also photochemically crosslinked to the 150-nucleotide SRP RNA fragment, providing the means to potentially identify portions of hexahistidine-SRP19 which are in close proximity to the RNA molecule.

Amino Acid Sequence↗

PubMed1997

Interaction of rice and human SRP19 polypeptides with signal recognition particle RNA.

The signal recognition particle (SRP) controls the transport of secretory proteins into and across lipid bilayers. SRP-like ribonucleoprotein complexes exist in all organisms, including plants. We characterized the rice SRP RNA and its primary RNA binding protein, SRP19. The secondary structure of the rice SRP RNA was similar to that found in other eukaryotes; however, as in other plant SRP RNAs, a GUUUCA hexamer sequence replaced the highly conserved GNRA-tetranucleotide loop motif at the apex of helix 8. The small domain of the rice SRP RNA was reduced considerably. Structurally, rice SRP19 lacked two small region that can be present in other SRP19 homologues. Conservative structure prediction and site-directed mutagenesis of rice and human SRP19 polypeptides indicated that binding to the SRP RNAs occurred via a loop that is present in the N-domain of both proteins. Rice SRP19 protein was able to form a stable complex with the rice SRP RNA in vitro. Furthermore, heterologous ribonucleoprotein complexes with components of the human SRP were assembled, thus confirming a high degree of structural and functional conservation between plant and mammalian SRP components.

Amino Acid Sequence↗

PubMed1997

Three-dimensional comparative modeling of RNA.

Comparative sequence analysis and ERNA-3D software were used to model the three-dimensional structure of the small domain of signal recognition particle RNA. RNA secondary structures were established by allowing only phylogenetically-supported base pairs. The folding of the RNA molecules was constrained further to include a well-supported pseudoknot. Helical sections were oriented coaxially where a continuous helical stack was formed in the RNA of another species. Finally, RNA helices were placed at distances that preserved the connectivity of the molecule with the smallest number of single-stranded nucleotide residues as identified from the aligned sequences. We show that the comparative three-dimensional structure modeling approach is an extremely powerful tool as it requires only a critical number of carefully aligned sequences.

Bacillus subtilis↗

PubMed1996

The uRNA database.

The uRNADB provides aligned, annotated and phylogenetically ordered sequences of the uRNAs. At the time of manuscript submission (August 1995) it contained sequences for U1 (78 sequences), U2 (80), U3 (20), U4 (38), U5 (37) and U6 (46). Additional uRNAs of interest will be available by the time you read this.

Animals↗

PubMed1996

The Signal Recognition Particle Database (SRPDB).

The Signal Recognition Particle Database (SRPDB) provides aligned SRP RNA and SRP protein sequences, annotated and phylogenetically ordered. The current release included 93 RNAs and 29 proteins representing SRP9, SRP14, SRP19, SRP21, SRP54, SRP68 and SRP72. The SRPDB can be downloaded and is accessible via the World Wide Web.

Computer Communication Networks↗