Biomedical subjects

J Wower

Publications and source records attributed to J Wower.

At least 19 recordsLinked to original sources

PubMed2001

tmRDB (tmRNA database).

The tmRNA database (tmRDB) is maintained at the University of Texas Health Science Center at Tyler, Texas, and accessible on the World Wide Web at the URL http://psyche.uthct.edu/dbs/tmRDB/tmRDB.++ +html. Mirror sites are located at Auburn University, Auburn, Alabama (http://www.ag.auburn.edu/mirror/tmRDB/) and the Institute of Biological Sciences, Aarhus, Denmark (http://www.bioinf.au. dk/tmRDB/). The tmRDB provides information and citation links about tmRNA, a molecule that combines functions of tRNA and mRNA in trans-translation. tmRNA is likely to be present in all bacteria and has been found in algae chloroplasts, the cyanelle of Cyanophora paradoxa and the mitochondrion of the flagellate Reclinomonas americana. This release adds 26 new sequences and corresponding predicted tmRNA-encoded tag peptides for a total of 86 tmRNAs, ordered alphabetically and phylogenetically. Secondary structures and three-dimensional models in PDB format for representative molecules are being made available. tmRNA alignments prove individual base pairs and are generated manually assisted by computational tools. The alignments with their corresponding structural annotation can be obtained in various formats, including a new column format designed to improve and simplify computational usability of the data.

Databases, Factual↗

PubMed2000

Transit of tRNA through the Escherichia coli ribosome. Cross-linking of the 3' end of tRNA to specific nucleotides of the 23 S ribosomal RNA at the A, P, and E sites.

When bound to Escherichia coli ribosomes and irradiated with near-UV light, various derivatives of yeast tRNA(Phe) containing 2-azidoadenosine at the 3' terminus form cross-links to 23 S rRNA and 50 S subunit proteins in a site-dependent manner. A and P site-bound tRNAs, whose 3' termini reside in the peptidyl transferase center, label primarily nucleotides U2506 and U2585 and protein L27. In contrast, E site-bound tRNA labels nucleotide C2422 and protein L33. The cross-linking patterns confirm the topographical separation of the peptidyl transferase center from the E site domain. The relative amounts of label incorporated into the universally conserved residues U2506 and U2585 depend on the occupancy of the A and P sites by different tRNA ligands and indicates that these nucleotides play a pivotal role in peptide transfer. In particular, the 3'-adenosine of the peptidyl-tRNA analogue, AcPhe-tRNA(Phe), remains in close contact with U2506 regardless of whether its anticodon is located in the A site or P site. Our findings, therefore, modify and extend the hybrid state model of tRNA-ribosome interaction. We show that the 3'-end of the deacylated tRNA that is formed after transpeptidation does not immediately progress to the E site but remains temporarily in the peptidyl transferase center. In addition, we demonstrate that the E site, defined by the labeling of nucleotide C2422 and protein L33, represents an intermediate state of binding that precedes the entry of deacylated tRNA into the F (final) site from which it dissociates into the cytoplasm.

Escherichia coli↗

PubMed2000

Binding and cross-linking of tmRNA to ribosomal protein S1, on and off the Escherichia coli ribosome.

UV irradiation of an in vitro translation mixture induced cross-linking of 4-thioU-substituted tmRNA to Escherichia coli ribosomes by forming covalent complexes with ribosomal protein S1 and 16S rRNA. In the absence of S1, tmRNA was unable to bind and label ribosomal components. Mobility assays on native gels demonstrated that protein S1 bound to tmRNA with an apparent binding constant of 1 x 10(8) M(-1). A mutant tmRNA, lacking the tag coding region and pseudoknots pk2, pk3 and pk4, did not compete with full-length tmRNA, indicating that this region is required for S1 binding. This was confirmed by identification of eight cross-linked nucleotides: U85, located before the resume codon of tmRNA; U105, in the mRNA portion of tmRNA; U172 in pK2; U198, U212, U230 and U240 in pk3; and U246, in the junction between pk3 and pk4. We concluded that ribosomal protein S1, in concert with the previously identified elongation factor EF-Tu and protein SmpB, plays an important role in tmRNA-mediated trans-translation by facilitating the binding of tmRNA to ribosomes and forming complexes with free tmRNA.

Bacterial Proteins↗

PubMed2000

Kinetic parameters for tmRNA binding to alanyl-tRNA synthetase and elongation factor Tu from Escherichia coli.

Aminoacylation and transportation of tmRNA to stalled ribosomes constitute prerequisite steps for trans-translation, a process facilitating the release of stalled ribosomes from 3' ends of truncated mRNAs and the degradation of incompletely synthesized proteins. Kinetic analysis of the aminoacylation of tmRNA indicates that tmRNA has both a lower affinity and a lower turnover number than cognate tRNA(Ala) for alanyl-tRNA synthetase, resulting in a 75-fold lower k(cat)/K(M) value. The association rate constant of Ala-tmRNA for elongation factor Tu in complex with GTP is about 150-fold lower than that of Ala-tRNA(Ala), whereas its dissocation rate constant is about 5-fold lower. These observations can be interpreted to suggest that additional factors facilitate tmRNA binding to ribosomes.

Acylation↗

PubMed2000

tmRDB (tmRNA database).

The tmRNA database (tmRDB) is maintained at the University of Texas Health Science Center at Tyler, Texas, and is accessible on the WWW at URL http://psyche.uthct.edu/dbs/tmRDB/tmRDB.++ +html. A tmRDB mirror site is located on the campus of Auburn University, Auburn, Alabama, reachable at the URL http://www.ag.auburn.edu/mirror/tmRDB/. Since April 1997, the tmRDB has provided sequences of tmRNA (previously called 10Sa RNA), a molecule present in most bacteria and some organelles. This release adds 17 new sequences for a total of 60 tmRNAs. Sequences and corresponding tmRNA-encoded tag peptides are tabulated in alphabetical and phylo-genetic order. The updated tmRNA alignment improves the secondary structures of known tmRNAs on the level of individual basepairs. tmRDB also provides an introduction to tmRNA function in trans-translation (with links to relevant literature), a limited number of tmRNA secondary structure diagrams, and numerous three-dimensional models generated interactively with the program ERNA-3D.

Databases, Factual↗

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↗

PubMed1998

Ribosomal protein L27 participates in both 50 S subunit assembly and the peptidyl transferase reaction.

Protein L27 has been implicated as a constituent of the peptidyl transferase center of the Escherichia coli 50 S ribosomal subunit by a variety of experimental observations. To define better the functional role of this protein, we constructed a strain in which the rpmA gene, which encodes L27, was replaced by a kanamycin resistance marker. The deletion mutant grows five to six times slower than the wild-type parent and is both cold- and temperature-sensitive. This phenotype is reversed when L27 is expressed from a plasmid-borne copy of the rpmA gene. Analysis of ribosomes from the L27-lacking strain revealed deficiencies in both the assembly and activity of the 50 S ribosomal subunits. Although functional 50 S subunits are formed in the mutant, an assembly "bottleneck" was evidenced by the accumulation of a prominent 40 S precursor to the 50 S subunit which was deficient in proteins L16, L20, and L21, as well as L27. In addition, the peptidyl transferase activity of 70 S ribosomes containing mutant 50 S subunits was determined to be three to four times lower than for wild-type ribosomes. Ribosomes lacking L27 were found to be impaired in the enzymatic binding of Phe-tRNAPhe to the A site, although the interaction of N-acetyl-Phe-tRNAPhe with the P site was largely unperturbed. We therefore infer that L27 contributes to peptide bond formation by facilitating the proper placement of the acceptor end of the A-site tRNA at the peptidyl transferase center.

Binding Sites↗

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↗

PubMed1994

Recombinant photoreactive tRNA molecules as probes for cross-linking studies.

Photoreactive tRNA derivatives have been used extensively for investigating the interaction of tRNA molecules with their ligands and substrates. Recombinant RNA technology facilitates the construction of such tRNA probes through site-specific incorporation of photoreactive nucleosides. The general strategy involves preparation of suitable tRNA fragments and their ligation either to a photoreactive nucleotide or to each other. tRNA fragments can be prepared by site-specific cleavage of native tRNAs, or synthesized by enzymatic and chemical means. A number of photoreactive nucleosides suitable for incorporation into tRNA are presently available. Joining of tRNA fragments is accomplished either by RNA ligase or by DNA ligase in the presence of a DNA splint. The application of this methodology to the study of tRNA binding sites on the ribosome is discussed, and a model of the tRNA-ribosome complex is presented.

Cross-Linking Reagents↗

PubMed1993

Mapping the central fold of tRNA2(fMet) in the P site of the Escherichia coli ribosome.

4-Thiouridine (s4U), a photoreactive analog of uridine, was randomly incorporated into tRNA2(fMet) precursor molecules by transcription with T7 RNA polymerase. The s4U-containing transcripts were trimmed at their 5'-ends with RNase P RNA to yield mature tRNA2(fMet). The photoreactive tRNA2(fMet) derivatives were aminoacylated and bound to the P site of 70S ribosomes from Escherichia coli in the presence of a poly(A,G,U) template. Irradiation of the complexes at 300 nm resulted in the covalent cross-linking of tRNA2(fMet) to ribosomal proteins and rRNAs within both the 50S and 30S subunits. The labeled proteins were identified as L1, L27, and S19. 50S-subunit proteins L1 and L27 were attached to nucleotide U17 or U17.1 within the D loop of tRNA2(fMet), whereas 30S-subunit protein S19 was cross-linked to nucleotide U47 in the variable loop. Both of these sites occur in or near the central fold of the tRNA. These results permit us to map the D loop of P site-bound tRNA to the region between the central protuberance and the L1 ridge on the 50S ribosomal subunit, while the variable loop can be placed above the cleft on the head of the 30S subunit.

Base Sequence↗

PubMed1993

Topography of the E site on the Escherichia coli ribosome.

Three photoreactive tRNA probes have been utilized in order to identify ribosomal components that are in contact with the aminoacyl acceptor end and the anticodon loop of tRNA bound to the E site of Escherichia coli ribosomes. Two of the probes were derivatives of E. coli tRNA(Phe) in which adenosines at positions 73 and 76 were replaced by 2-azidoadenosine. The third probe was derived from yeast tRNA(Phe) by substituting wyosine at position 37 with 2-azidoadenosine. Despite the modifications, all of the photoreactive tRNA species were able to bind to the E site of E. coli ribosomes programmed with poly(A) and, upon irradiation, formed covalent adducts with the ribosomal subunits. The tRNA(Phe) probes modified at or near the 3' terminus exclusively labeled protein L33 in the 50S subunit. The tRNA(Phe) derivative containing 2-azidoadenosine within the anticodon loop became cross-linked to protein S11 as well as to a segment of the 16S rRNA encompassing the 3'-terminal 30 nucleotides. We have located the two extremities of the E site-bound tRNA on the ribosomal subunits according to the positions of L33, S11 and the 3' end of 16S rRNA defined by immune electron microscopy. Our results demonstrate conclusively that the E site is topographically distinct from either the P site or the A site, and that it is located alongside the P site as expected for the tRNA exit site.

Adenosine↗

PubMed1992

Photochemical cross-linking of the anticodon loop of yeast tRNA(Phe) to 30S-subunit protein S7 at the ribosomal A and P sites.

Yeast tRNA(Phe), containing the photoreactive nucleoside 2-azidoadenosine at position 37 within the anticodon loop, has been cross-linked to the aminoacyl-tRNA (A) and peptidyl-tRNA (P) binding sites of the Escherichia coli ribosome. The 30S subunit was exclusively labeled in each case, and cross-linking occurred to both protein and 16S rRNA. Electrophoretic and immunological analyses demonstrated that S7 was the only 30S-subunit protein covalently attached to the tRNA. However, digestion of the A and P site-labeled S7 with trypsin revealed a unique pattern of cross-linked peptide(s) at each site. Thus, while the anticodon loop of tRNA is in close proximity to protein S7 at both the A and P sites, it neighbors a different portion of the protein molecule in each. The placement of the aminoacyl- and peptidyl-tRNA binding sites is discussed in relationship to recent models of the 30S ribosomal subunit.

Anticodon↗

PubMed1990

Probing tRNA binding sites on the Escherichia coli 30 S ribosomal subunit with photoreactive analogs of the anticodon arm.

Two analogs of the anticodon arm of yeast tRNAPhe (residues 28-43), in which G43 was replaced by the photoreactive nucleosides 2-azidoadenosine and 8-azidoadenosine, have been used to create 'zero-length' cross-links to ribosomal components at the peptidyl-tRNA binding site (P site) of 30 S subunits from the Escherichia coli ribosome. To prepare the analogs, 2-azidoadenosine and 8-azidoadenosine bisphosphates were first ligated to the 3' end of the anticodon-containing dodecanucleotide ACmUGmAAYA psi m5CUG from yeast tRNAPhe. The trinucleotide CAG was then joined to the 5' end of the resulting tridecanucleotide in a subsequent ligation. Both analogs bound to poly(U)-programmed 30 S subunits with affinities similar to that of the unmodified anticodon arm from yeast tRNAPhe. Irradiation of noncovalent complexes containing the photolabile analogs, poly(U) and 30 S ribosomal subunits with 300 nm light led to the covalent attachment of the anticodon arms to proteins S13 and S19. Further analysis revealed that S13 accounted for about 80%, and S19 for about 20%, of the cross-linked material. Labeling of these two proteins with 'zero-length' cross-linking probes provides useful information about the location and orientation of P site-bound tRNA on the ribosome and permits a test of recently proposed models of the three-dimensional structure of the 30 S subunit.

Adenosine↗

PubMed1989

Preparation of 2-azidoadenosine 3',5'-[5'-32P]bisphosphate for incorporation into transfer RNA. Photoaffinity labeling of Escherichia coli ribosomes.

2-Azidoadenosine was synthesized from 2-chloroadenosine by sequential reaction with hydrazine and nitrous acid and then bisphosphorylated with pyrophosphoryl chloride to form 2-azidoadenosine 3',5'-bisphosphate. The bisphosphate was labeled in the 5'-position using the exchange reaction catalyzed by T4 polynucleotide kinase in the presence of [gamma-32P]ATP. Polynucleotide kinase from a T4 mutant which lacks 3'-phosphatase activity (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) was required to facilitate this reaction. 2-Azidoadenosine 3',5'-[5'-32P]bisphosphate can serve as an efficient donor in the T4 RNA ligase reaction and can replace the 3'-terminal adenosine of yeast tRNAPhe with little effect on the amino acid acceptor activity of the tRNA. In addition, we show that the modified tRNAPhe derivative can be photochemically cross-linked to the Escherichia coli ribosome.

Adenosine Diphosphate↗

PubMed1989

Photochemical labeling of bovine pancreatic ribonuclease A with 8-azidoadenosine 3',5'-bisphosphate.

A simple method has been developed for the preparation of 5'-32P-labeled 8-azidoadenosine 3',5'-bisphosphate (p8N3Ap) for use in photoaffinity labeling studies. Irradiation of a complex between p8N3Ap and bovine pancreatic ribonuclease A (RNase A) with light of 300-350 nm led to the covalent attachment of the nucleotide to the enzyme. RNase A could also be labeled in the dark with prephotolyzed p8N3Ap. In either case, the nucleotide reacted with the same tryptic peptide, encompassing amino acids 67-85 of the protein. The site of labeling was determined to be either Thr-78 or Thr-82, both of which are close to or at the pyrimidine binding site of the enzyme. This result is consistent with recent nuclear magnetic resonance and X-ray studies which indicate that 8-substituted adenine nucleotides interact with the pyrimidine binding site of RNase A.

Adenosine Diphosphate↗

PubMed1989

Labeling the peptidyltransferase center of the Escherichia coli ribosome with photoreactive tRNA(Phe) derivatives containing azidoadenosine at the 3' end of the acceptor arm: a model of the tRNA-ribosome complex.

Photoreactive derivatives of yeast tRNA(Phe) containing 2-azidoadenosine (2N3A) at position 73 or 76 have been crosslinked to the peptidyl site of Escherichia coli ribosomes. Covalent tRNA-ribosome attachment was dependent upon the replacement of adenosine by 2N3A in the tRNA, irradiation with 300-nm light, and the presence of poly(U). In all cases, the modified tRNAs became crosslinked exclusively to 50S ribosomal subunits. While the tRNA derivative containing 2N3A at position 73 labeled only protein L27, that containing 2N3A at position 76 labeled proteins L15, L16, and L27 as well as a segment of the 23S rRNA. The site of crosslinking in the rRNA was identified as guanosine-1945, which lies within a highly conserved sequence adjacent to a number of modified bases and has not until now been identified at the peptidyltransferase center. On the basis of these results, and previously reported crosslinks from tRNA containing 8-azidoadenosine in the 3'-terminal -A-C-C-A sequence [Wower, J., Hixson, S. S. & Zimmermann, R. A. (1988) Biochemistry 27, 8114-8121], we propose a model for the arrangement of tRNA molecules at the peptidyl and aminoacyl sites that is consistent with most of the information available about the location of the peptidyltransferase center and the decoding domain of the E. coli ribosome.

Acyltransferases↗

PubMed1988

Photochemical cross-linking of yeast tRNA(Phe) containing 8-azidoadenosine at positions 73 and 76 to the Escherichia coli ribosome.

The 3'-terminal -A-C-C-A sequence of yeast tRNA(Phe) has been modified by replacing either adenosine-73 or adenosine-76 with the photoreactive analogue 8-azidoadenosine (8N3A). The incorporation of 8N3A into tRNA(Phe) was accomplished by ligation of 8-azidoadenosine 3',5'-bisphosphate to the 3' end of tRNA molecules which were shortened by either one or four nucleotides. Replacement of the 3'-terminal A76 with 8N3A completely blocked aminoacylation of the tRNA. In contrast, the replacement of A73 with 8N3A has virtually no effect on the aminoacylation of tRNA(Phe). Neither substitution hindered binding of the modified tRNAs to Escherichia coli ribosomes in the presence of poly(U). Photoreactive tRNA derivatives bound noncovalently to the ribosomal P site were cross-linked to the 50S subunit upon irradiation at 300 nm. Nonaminoacylated tRNA(Phe) containing 8N3A at either position 73 or position 76 cross-linked exclusively to protein L27. When N-acetylphenylalanyl-tRNA(Phe) containing 8N3A at position 73 was bound to the P site and irradiated, 23S rRNA was the main ribosomal component labeled, while smaller amounts of the tRNA were cross-linked to proteins L27 and L2. Differences in the labeling pattern of nonaminoacylated and aminoacylated tRNA(Phe) containing 8N3A in position 73 suggest that the aminoacyl moiety may play an important role in the proper positioning of the 3' end of tRNA in the ribosomal P site. More generally, the results demonstrate the utility of 8N3A-substituted tRNA probes for the specific labeling of ribosomal components at the peptidyltransferase center.

Adenosine↗