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A Bakin

Publications and source records attributed to A Bakin.

14 recordsLinked to original sources
PubMed2001

Transforming growth factor-beta1 mediates epithelial to mesenchymal transdifferentiation through a RhoA-dependent mechanism.

Transforming growth factor-beta1 (TGF-beta) can be tumor suppressive, but it can also enhance tumor progression by stimulating the complex process of epithelial-to-mesenchymal transdifferentiaion (EMT). The signaling pathway(s) that regulate EMT in response to TGF-beta are not well understood. We demonstrate the acquisition of a fibroblastoid morphology, increased N-cadherin expression, loss of junctional E-cadherin localization, and increased cellular motility as markers for TGF-beta-induced EMT. The expression of a dominant-negative Smad3 or the expression of Smad7 to levels that block growth inhibition and transcriptional responses to TGF-beta do not inhibit mesenchymal differentiation of mammary epithelial cells. In contrast, we show that TGF-beta rapidly activates RhoA in epithelial cells, and that blocking RhoA or its downstream target p160(ROCK), by the expression of dominant-negative mutants, inhibited TGF-beta-mediated EMT. The data suggest that TGF-beta rapidly activates RhoA-dependent signaling pathways to induce stress fiber formation and mesenchymal characteristics.

Animals↗
PubMed2000

Isolation and properties of Escherichia coli 23S-RNA pseudouridine 1911, 1915, 1917 synthase (RluD).

All nine pseudouridine (psi) residues in Escherichia coli 23S RNA are in or very near the peptidyl transfer centre (PTC) of the ribosome. Five psi synthases catalyze synthesis of these nine psi's. Deletion of the gene for one psi synthase, RluD, which directs synthesis of three closely clustered psi's in the decoding site of the PTC, has a profound negative impact on cell growth. We describe the isolation, without amplification from a cloned coding element, of the triple-site modifying enzyme, RluD, the N-terminal sequence of which has been used to clone and express the corresponding gene, rluD. Unlike "expressed" RluD, which so far has not been shown to modify one (1911) of the three closely clustered sites (1911, 1915, 1917), "natural" RluD modifies all three sites; and unlike another pai synthase, RluA, natural RluD has greatly expanded modifying activity at low Mg concentrations. These properties of the expressed and natural forms of RluD are discussed.

Amino Acid Sequence↗
PubMed1997

Mapping to nucleotide resolution of pseudouridine residues in large subunit ribosomal RNAs from representative eukaryotes, prokaryotes, archaebacteria, mitochondria and chloroplasts.

The pseudouridine (psi) residues present in the high molecular mass RNA from the large ribosomal subunit (LSU) have been sequenced from representative species of the eukaryotes, prokaryotes and archaebacteria, and from mitochondrial and chloroplast organelles. Ribosomes from Bacillus subtilis, Halobacter halobium, Drosphilia melanogaster, Mus musculus, Homo sapiens, mitochondria of M. musculus, H. sapiens and Trypanosoma brucei, and Zea mays chloroplasts were examined, resulting in the exact localization of 190 psi residues. The number of psi residues per RNA varied from one in the mitochondrial RNAs to 57 in the cytoplasmic LSU RNA of D. melanogaster and M. musculus. Despite this, all of the psi residues were found in three domains, II, IV and V. All three are at or have been linked to the peptidyl transferase center according to the literature. Comparison of the sites for psi among the species examined revealed four conserved or semi-conserved segments. One is the region 1911 to 1917, which contains three psi or modified psi in almost all species examined. This site is also juxtaposed to the decoding site of the 30 S subunit in the 70 S ribosome and has been implicated in the fidelity of codon recognition. Three additional sites were at the peptidyl transferase center itself. The juxtaposition of the conserved sites for psi with the two important functions of the ribosome, codon recognition and peptide bond formation, implies an important role for psi in ribosome function. We report some new putative modified nucleosides in LSU RNAs as detected by reverse transcription, correct a segment of the sequence of Z. mays chloroplasts and D. melanogaster LSU RNA, correlate the secondary structural context for all known psi residues in ribosomal RNA, and compare the sites for psi with those known for methylated nucleosides in H. sapiens.

Animals↗
PubMed1995

Mapping of the 13 pseudouridine residues in Saccharomyces cerevisiae small subunit ribosomal RNA to nucleotide resolution.

The number and location of all of the pseudouridine (phi) residues in Saccharomyces cerevisiae small subunit (SSU) ribosomal RNA have been determined by a reverse transcriptase sequencing method [Bakin, A. and Ofengand, J., 1993, Biochemistry, 32, 9754-9762]. Thirteen residues were found in addition to the previously described m1acp3 phi 1189. The residues were scattered throughout the molecule with three being in expansion segments. No phi was found in the three highly conserved single-stranded sequence elements common to all SSU RNAs. Specifically, phi 563, the analog of phi 516 (Escherichia coli) and phi 517 (Bacillus subtilis) were not found. Eight of the phi were located identically to those in mammalian SSU RNA and three were near to mammalian phi residues in the secondary structure. There was no discernible correlation between the sites for phi and the known locations of the methylated nucleosides as exists in large subunit (LSU) RNAs. Comparison of the structural context in which phi was found in SSU RNA with that in LSU RNA showed a differential bias suggestive of possible different roles for phi in the two rRNAs. This work also identified the locations of three putative new modified bases in SSU rRNA, and revealed 15 sequence differences between the yeast strain used here and the reported sequence.

Base Sequence↗
PubMed1995

Purification, cloning, and properties of the 16S RNA pseudouridine 516 synthase from Escherichia coli.

Pseudouridine (psi) is commonly found in both small and large subunit ribosomal RNAs of prokaryotes and eukaryotes. In Escherichia coli small subunit RNA, there is only one psi, at position 516, in a region of the RNA known to be involved in codon recognition [Bakin et al. (1994) Nucleic Acids Res. 22, 3681-3684]. To assess the function of this single psi residue, the enzyme catalyzing its formation was purified and cloned. The enzyme contains 231 amino acids and has a calculated molecular mass of 25,836 Da. It converts U516 in E. coli 16S RNA transcripts into psi but does not modify any other position in this RNA. It does not react with free unmodified 16S RNA at all, and only poorly with 30S particles containing unmodified RNA. The preferred substrate is an RNA fragment from residues 1 to 678 which has been complexed with 30S ribosomal proteins. The yield varied from 0.6 to 1.0 mol of psi/mol of RNA, depending on the preparation. Free RNA(1-678) was inactive, as was RNA(1-526) and the RNP particle made from it. 23S RNA and tRNAVal transcripts were also inactive. These results suggest that psi formation in vivo occurs at an intermediate stage of 30S assembly. The gene is located at 47.1 min immediately 5' to, and oriented in the same direction as, the bicyclomycin resistance gene. The gene was cloned behind a (His)6 leader for affinity purification. Virtually all of the overexpressed protein was found in inclusion bodies but could be purified to homogeneity on a Ni2+(-) containing resin. Over 200 mg of pure protein could be obtained from a liter of cell culture. Amino acid sequence comparison revealed the existence of a gene in Bacillus subtilis with a similar sequence, and psi sequence analysis established that B. subtilis has the equivalent of psi 516 in its small subunit rRNA. On the other hand, no common sequence motifs could be detected among this enzyme and the two tRNA psi synthases which have been cloned up to now.

Amino Acid Sequence↗
PubMed1995

Purification, cloning, and properties of the tRNA psi 55 synthase from Escherichia coli.

tRNA pseudouridine 55 (psi 55) synthase, the enzyme that is specific for the conversion of U55 to psi 55 in the m5U psi CG loop in most tRNAs, has been purified from Escherichia coli and cloned. On SDS gels, a single polypeptide chain with a mass of 39.7 kDa was found. The gene is a previously described open reading frame, p35, located at 68.86 min on the E. coli chromosome between the infB and rpsO genes. The proposed name for this gene is truB. There is very little protein sequence homology between the truB gene product and the hisT (truA) product, which forms psi in the anticodon arm of tRNAs. However, there was high homology with a fragment of a Bacillus subtilis gene that may produce the analogous enzyme in that species. The cloned gene was fused to a 5'-leader coding for a (His)6 tract, and the protein was overexpressed > 400-fold in E. coli. The recombinant protein was purified to homogeneity in one step from a crude cell extract by affinity chromatography using a Ni(2+)-containing matrix. The SDS mass of the recombinant protein was 41.5 kDa, whereas that calculated from the gene was 37.3. The recombinant protein was specific for U55 in tRNA transcripts and reacted neither at other sites for psi in such transcripts nor with transcripts of 16S or 23S ribosomal RNA or subfragments. The enzyme did not require either a renatured RNA structure or Mg2+, and prior formation of m5U was not required. Stoichiometric formation of psi occurred with no requirement for an external source of energy, indicating that psi synthesis is thermodynamically favored.

Amino Acid Sequence↗
PubMed1995

A dual-specificity pseudouridine synthase: an Escherichia coli synthase purified and cloned on the basis of its specificity for psi 746 in 23S RNA is also specific for psi 32 in tRNA(phe).

An Escherichia coli pseudouridine (psi) synthase, which forms both psi 746 in E. coli 23S ribosomal RNA and psi 32 in tRNA(Phe), has been isolated and cloned. The enzyme contains 219 amino acids and has a calculated MW of 24,432 Da. Amino acid sequence comparison with the three other psi synthases that have been cloned to date, two for tRNA and one for 16S RNA, did not reveal any common sequence motifs, despite the catalysis of a common reaction. The gene was cloned behind a (His)6 leader for affinity purification. Upon overexpression, most of the enzyme remained soluble in the cell cytoplasm and could be purified to homogeneity on a Ni(2+)-containing resin. The enzyme reacted with both full-length 23S RNA or a fragment from residues 1-847, forming 1 mol psi/mol RNA at position 746, a normal site for psi. The enzyme has no dependence on Mg2+. The same yield was obtained in 1 mM EDTA as in 10 mM Mg2+, and the rate was faster in EDTA than in Mg2+. Full-length 16S RNA or fragments 1-526 or 1-678, as well as tRNA(Val) transcripts, were not modified in either EDTA or Mg2+. tRNA(Phe) transcripts, however, were modified with a yield of 1 mol psi/mol transcript at a rate in EDTA like that of 23S RNA. Sequencing showed all of the psi to be at position 32, a normal site for psi in this tRNA. Both 23S rRNA psi 746 and tRNA psi 32 occur in single-stranded segments of the same sequence, psi UGAAAA, closed by a stem. Therefore, this synthase may require for recognition only a short stretch of primary sequence 3' to the site of pseudouridylation. This is the first example of a dual-specificity modifying enzyme for RNA, that is, one which is specific for a single site in one RNA, and equally site-specific in a second class of RNA. The essentiality of these psi residues can now be assessed by disruption of the synthase gene.

Amino Acid Sequence↗
PubMed1994

Clustering of pseudouridine residues around the peptidyltransferase center of yeast cytoplasmic and mitochondrial ribosomes.

Analysis of the high molecular weight RNAs of the larger ribosomal subunit of Saccharomyces cerevisiae cytoplasm and mitochondria by a new method [Bakin, A., & Ofengand, J. (1993) Biochemistry 32, 9754-9762] has for the first time located all of the pseudouridine residues present in these two RNAs. Thirty pseudouridines were found in the cytoplasmic RNA, and one was found in the mitochondrial RNA. The 30 cytoplasmic RNA pseudouridines were clustered in three regions of the RNA known to be at or near the peptidyltransferase center. The single pseudouridine in yeast mitochondrial rRNA at position 2819 was also located at the peptidyltransferase center. The localization of pseudouridines at or near the peptidyltransferase center in both cytoplasmic and mitochondrial ribosomes implies a functional role for pseudouridine in peptide bond formation. A correlation was shown to exist between the locations of the pseudouridines determined in this work and the positions of the methylated nucleotides (both 2'-OCH3 and base-methylated) determined previously by others. In addition, this work has tentatively identified the locations of two previously unknown ribothymidine residues, at positions 955 and 2920 in the cytoplasmic rRNA.

Base Sequence↗
PubMed1994

The single pseudouridine residue in Escherichia coli 16S RNA is located at position 516.

The number and location of pseudouridine residues in Escherichia coli 16S ribosomal RNA has been determined by a combination of direct and indirect methods. Only one residue was found, at position 516. This site is at the 5'-end of one of the three most highly conserved long sequences of this RNA molecule. A number of experimental findings have strongly implicated this loop in the fidelity of codon recognition by A-site bound tRNA. By virtue of its location, we suggest that psi 516 may also play a role in maintaining the fidelity of protein synthesis.

Base Sequence↗
PubMed1993

Four newly located pseudouridylate residues in Escherichia coli 23S ribosomal RNA are all at the peptidyltransferase center: analysis by the application of a new sequencing technique.

A new technique has been developed for the facile location of pseudouridylate (psi) residues in any RNA molecule. The method uses two known modification procedures which in combination uniquely identify U residues which have been converted into psi. The first procedure involves reaction of all U-like and G-like residues with N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide p-tosylate (CMC), followed by alkaline removal of all CMC groups except those linked to the N3 of psi. This stops reverse transcription, resulting in a gel band which identifies the U residue. The second procedure is uridine-specific hydrazinolysis which cleaves the RNA chain at all U residues and produces a gel band upon reverse transcription. psi residues, being resistant to hydrazinolysis, are not cleaved and do not stop reverse transcription. This leads to the absence of a band at psi residues. The combined method can also distinguish psi from 5-methyluridine, 4-thiouridine, uridine-5-oxyacetic acid, and 2-thio-5-methylaminomethyluridine as shown by treating rRNA and tRNA species known to contain these modified bases at defined sites. By this procedure, four new sites for psi in Escherichia coli 23S RNA were discovered, and one was disproven. The four new sites are at positions 2457, 2504, 2580, and 2605. The erroneous site is at position 2555. These four new psi residues, which are all in or within 2-3 residues of the peptidyltransferase ring, are thus in a position to play a functional and/or structural role at the peptidyltransferase center.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1993

Functional effects of a G to U base change at position 530 in a highly conserved loop of Escherichia coli 16S RNA.

Any base change at position 530 introduced into Escherichia coli on a multicopy plasmid leads to cell death [Powers & Noller (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1042-1046]. It was suggested that these mutants cannot carry out chain elongation. To define more precisely the function of base 530, we have studied ribosomes in which G530 was mutated to U530. In vivo, U530 16S rRNA was incorporated into 30S subunits and could combine with 50S to make 70S ribosomes. 16S rRNA in vitro transcripts containing U530 were assembled into 30S ribosomes, and their activity was tested in defined steps of protein synthesis. Mutant 30S ribosomes were as active as wild-type in poly(U)-dependent poly(Phe) synthesis, P- and A-site tRNA binding, and 30S initiation complex formation. 30S initiation complexes, in the presence of 50S, could react with puromycin like the wild-type. The rate, extent, and position of cross-linking of AcVal-tRNA in the P site to 16S RNA were identical in mutant and wild-type ribosomes. Although there appeared to be no defect in 70S initiation complex formation or in direct A-site binding of Phe-tRNA dependent on poly(U), U530 30S ribosomes were nevertheless defective in carrying out synthesis of fMet-Val dipeptide using natural mRNA. Mutant 30S ribosomes were also refractory to streptomycin-induced misreading although no misreading was observed in its absence.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1992

Interaction between the two conserved single-stranded regions at the decoding site of small subunit ribosomal RNA is essential for ribosome function.

Formation of the tertiary base pair G1401:C1501, which brings together two universally present and highly sequence-conserved single-stranded segments of small subunit ribosomal RNA, is essential for ribosome function. It was previously reported that mutation of G1401 inactivated all in vitro functions of the ribosome [Cunningham et al. (1992) Biochemistry 31, 7629-7637]. Here we show that mutation of C1501 to G was equally inactivating but that the double mutant C1401:G1501 with the base pair reversed had virtually full activity for tRNA binding to the P, A, and I sites and for peptide bond formation. Initiation-dependent formation of the first peptide bond remained 70-85% inhibited, despite full 70S initiation complex formation ability as evidenced by the ability to form fMET-puromycin. These results suggest that the defect in formation of the first peptide bond lies in filling the initial A site, Ai, rather than the subsequent elongation A sites, Ae. An increased mobility around the anticodon was detected by UV cross-linking of the anticodon of P-site-bound tRNA to C1399 as well as to the expected C1400. These findings provide the first experimental evidence for the existence of the G1401:C1501 base pair and show that this base pair, located at the decoding site, is essential for function. The structural implications of tertiary base pair formation are discussed.

Base Sequence↗
PubMedDate not supplied

The pseudouridine residues of ribosomal RNA.

Pseudouridine (psi), the most common single modified nucleoside in ribosomal RNA, has been positioned in the small subunit (SSU) and large subunit (LSU) RNAs of a number of representative species. Most of the information has been obtained by application of a rapid primed reverse transcriptase sequencing technique. The locations of these psi residues have been compared. Many sites for psi are the same among species, but others are distinct. In general, the percentage psi in multicellular eukaryotes is greater than in prokaryotes. In LSU RNA, the psi residues are strongly clustered in three domains, all of which are near or connected to the peptidyl transferase center. There is no apparent clustering of psi in SSU RNA. The psi sites in LSU RNA overlap those for the methylated nucleosides, but this is not the case in SSU RNA. There are 265 psi sites known to nucleotide resolution, of which 246 are in defined secondary structures, and 112 of these are in nonidentical structural contexts. All 246 psi sites can be classified into five structural types. Two Escherichia coli psi synthases have been cloned and characterized, one for psi 516 in SSU RNA and one for psi 746 in LSU RNA. The psi 746 synthase recognizes free RNA, but the psi 516 enzyme requires an intermediate RNP particle. Possible functional roles for psi in the ribosome are discussed.

Base Sequence↗