Search PubMed⌕ Search

Biomedical subjects

J Wower

Publications and source records attributed to J Wower.

30 records · Page 2Linked to original sources
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↗
PubMed1983

Identification of tyrosine residues that are susceptible to lactoperoxidase-catalyzed iodination on the surface of Escherichia coli 50s ribosomal subunits or 70s ribosomes.

Further to our studies on the Escherichia coli 30S ribosomal subunit, the detailed surface topography of both 50S subunits and 70S ribosomes has been investigated by using iodination catalyzed by immobilized lactoperoxidase as the surface probe. In the 50S subunit, only proteins L2, L5, L10, and L11 were iodinated to a significant and reproducible extent. The targets of iodination were identified, after isolation of the individual iodinated proteins, and were as follows: in protein L2 (271 amino acids), tyrosine-102 and -160; in protein L5 (178 amino acids), tyrosine-142; in protein L10 (165 amino acids), tyrosine-132; in protein L11 (142 amino acids), tyrosine-7 and -61. In the 70S ribosome, only protein L5 was still iodinated to a significant extent from the 50S subunit, whereas in the 30S subunit the same spectrum of iodinated proteins was observed as that from iodinated isolated 30S subunits, with the exception that S21 was no longer present.

Escherichia coli↗
PubMed1983

Identification of tyrosine residues that are susceptible to lactoperoxidase-catalyzed iodination on the surface of Escherichia coli 30S ribosomal subunit.

The detailed surface topography of the Escherichia coli 30S ribosomal subunit has been investigated, with iodination catalyzed by immobilized lactoperoxidase as the surface probe. Under mild conditions, only proteins S3, S7, S9, S18, and S21 were iodinated to a significant and reproducible extent. These proteins were isolated from the iodinated subunits, and in each case, the individual tyrosine residues that had reacted were identified by standard protein sequencing techniques. The targets of iodination that could be positively established were as follows: in protein S3 (232 amino acids), the tyrosines at positions 167 and 192; in S7 (153 amino acids), tyrosines 84 and 152; in S9 (128 amino acids), tyrosine 89; in S18 (74 amino acids), tyrosine 3 (tentative); in S21 (70 amino acids), tyrosines 37 and 70. The results represent part of a broader program to investigate ribosomal topography at the amino acid-nucleotide level.

Amino Acid Sequence↗
PubMed1981

The use of 2-iminothiolane as an RNA-protein cross-linking agent in Escherichia coli ribosomes, and the localisation on 23S RNA of sites cross-linked to proteins L4, L6, L21, L23, L27 and L29.

When E. coli ribosomal subunits are reacted with 2-iminothiolane and then subjected to a mild ultraviolet irradiation, an RNA-protein cross-linking reaction occurs. About 5% of the total protein in each subunit becomes cross-linked to the RNA, and a specific sub-set of proteins is involved in the reaction. In the case of the 50S subunit, the sites of cross-linking to the 23S RNA have been determined for six of these proteins: protein L4 is cross-linked within an oligonucleotide comprising positions 613-617 in the 23S sequence, L6 within positions 2473-2481, L21 within positions 540-548, L23 within positions 137-141, L27 within positions 2332-2337 and L29 within positions 99-107.

Bacterial Proteins↗
PubMedDate not supplied

A consonant model of the tRNA-ribosome complex during the elongation cycle of translation.

Chemical and photochemical affinity techniques have been used extensively to determine the positions of the tRNA binding sites on the Escherichia coli ribosome. Recent advances in our understanding of ribosome structure and function prompted us to critically review the data that have accumulated on tRNA-ribosome cross-links. As a result, we propose a new model of the tRNA-ribosome complex that accounts for nearly all of the pertinent evidence.

Binding Sites↗
PubMedDate not supplied

Synthesis of 2,6-diazido-9-(beta-D-ribofuranosyl)purine 3',5'-bisphosphate: incorporation into transfer RNA and photochemical labeling of Escherichia coli ribosomes.

2,6-Diazido-9-(beta-D-ribofuranosyl)purine was prepared by the reaction of 2,6-dichloro-9-(beta-D-ribofuranosyl)purine with sodium azide. The nucleoside was bisphosphorylated with pyrophosphoryl chloride to form 2,6-diazido-9-(beta-D-ribofuranosyl)purine 3',5'-bisphosphate. This product was labeled with 32P using T4 polynucleotide kinase to exchange the 5' phosphate with the gamma phosphate of [gamma-32P]ATP. When yeast tRNA(Phe) containing 2,6-diazido-9-(beta-D-ribofuranosyl)purine at the 3' terminus was bound to the P site of the Escherichia coli ribosome in the presence of poly(U) and irradiated with 300-nm light, the photoreactive tRNA derivative became cross-linked exclusively to the 50S subunit. The label was attached to proteins L27 and L33 as well as to the 23S rRNA.

Adenosine↗
PubMedDate not supplied

Peptidyl transferase and beyond.

The peptidyl transferase center of the Escherichia coli ribosome encompasses a number of 50S-subunit proteins as well as several specific segments of the 23S rRNA. Although our knowledge of the role that both ribosomal proteins and 23S rRNA play in peptide bond formation has steadily increased, the location, organization, and molecular structure of the peptidyl transferase center remain poorly defined. Over the past 10 years, we have developed a variety of photoaffinity reagents and strategies for investigating the topography of tRNA binding sites on the ribosome. In particular, we have used the photoreactive tRNA probes to delineate ribosomal components in proximity to the 3' end of tRNA at the A, P, and E sites. In this article, we describe recent experiments from our laboratory which focus on the identification of segments of the 23S rRNA at or near the peptidyl transferase center and on the functional role of L27, the 50S-subunit protein most frequently labeled from the acceptor end of A- and P-site tRNAs. In addition, we discuss how these results contribute to a better understanding of the structure, organization, and function of the peptidyl transferase center.

Binding Sites↗