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W Keller

Publications and source records attributed to W Keller.

At least 37 records · Page 2Linked to original sources
PubMed1999

An adenosine deaminase that generates inosine at the wobble position of tRNAs.

Several transfer RNAs (tRNAs) contain inosine (I) at the first position of their anticodon (position 34); this modification is thought to enlarge the codon recognition capacity during protein synthesis. The tRNA-specific adenosine deaminase of Saccharomyces cerevisiae that forms I(34) in tRNAs is described. The heterodimeric enzyme consists of two sequence-related subunits (Tad2p/ADAT2 and Tad3p/ADAT3), both of which contain cytidine deaminase (CDA) motifs. Each subunit is encoded by an essential gene (TAD2 and TAD3), indicating that I(34) is an indispensable base modification in elongating tRNAs. These results provide an evolutionary link between the CDA superfamily and RNA-dependent adenosine deaminases (ADARs/ADATs).

Adenosine Deaminase↗
PubMed1999

Editing of messenger RNA precursors and of tRNAs by adenosine to inosine conversion.

The double-stranded RNA-specific adenosine deaminases ADAR1 and ADAR2 convert adenosine (A) residues to inosine (I) in messenger RNA precursors (pre-mRNA). Their main physiological substrates are pre-mRNAs encoding subunits of ionotropic glutamate receptors or serotonin receptors in the brain. ADAR1 and ADAR2 have similar sequence features, including double-stranded RNA binding domains (dsRBDs) and a deaminase domain. The tRNA-specific adenosine deaminases Tad1p and Tad2p/Tad3p modify A 37 in tRNA-Ala1 of eukaryotes and the first nucleotide of the anticodon (A 34) of several bacterial and eukaryotic tRNAs, respectively. Tad1p is related to ADAR1 and ADAR2 throughout its sequence but lacks dsRBDs. Tad1p could be the ancestor of ADAR1 and ADAR2. The deaminase domains of ADAR1, ADAR2 and Tad1p are very similar and resemble the active site domains of cytosine/cytidine deaminases.

Adenosine↗
PubMed1999

mRNA polyadenylation and its coupling to other RNA processing reactions and to transcription.

Eukaryotic mRNA precursors are processed at their 3' ends by a coupled cleavage/polyadenylation reaction. In recent years, most of the factors involved in 3'-end processing have been identified and evidence has been presented for the coupling of mRNA 3'-end formation to capping, splicing and transcription. These links are important for the quality control of the mRNA during synthesis.

Animals↗
PubMed1999

Mapping of ATP binding regions in poly(A) polymerases by photoaffinity labeling and by mutational analysis identifies a domain conserved in many nucleotidyltransferases.

We have identified regions in poly(A) polymerases that interact with ATP. Conditions were established for efficient cross-linking of recombinant bovine and yeast poly(A) polymerases to 8-azido-ATP. Mn2+ strongly stimulated this reaction due to a 50-fold lower Ki for 8-azido-ATP in the presence of Mn2+. Mutations of the highly conserved Asp residues 113, 115, and 167, critical for metal binding in the catalytic domain of bovine poly(A) polymerase, led to a strong reduction of cross-linking efficiency, and Mn2+ no longer stimulated the reaction. Sites of 8-azido-ATP cross-linking were mapped in different poly(A) polymerases by CNBr-cleavage and analysis of tryptic peptides by mass spectroscopy. The main cross-link in Schizosaccharomyces pombe poly(A) polymerase could be assigned to the peptide DLELSDNNLLK (amino acids 167-177). Database searches with sequences surrounding the cross-link site detected significant homologies to other nucleotidyltransferase families, suggesting a conservation of the nucleotide-binding fold among these families of enzymes. Mutations in the region of the "helical turn motif" (a domain binding the triphosphate moiety of the nucleotide) and in the suspected nucleotide-binding helix of bovine poly(A) polymerase impaired ATP binding and catalysis. The results indicate that ATP is bound in part by the helical turn motif and in part by a region that may be a structural analog to the fingers domain found in many polymerases.

Adenosine Triphosphate↗
PubMed1999

Thermodynamic properties and DNA binding of the ParD protein from the broad host-range plasmid RK2/RP4 killing system.

ParD is a small, acidic protein from the partitioning system of the plasmid RK2/RP4. The ParD protein exhibits specific DNA binding activity and, as the antidote component of a toxin-antidote plasmid addiction system, ParD forms a tight complex in solution with its toxin antagonist, the ParE protein. Unopposed ParE acts as a toxin that causes growth retardation and killing of plasmid cured cells. ParD negatively autoregulates its expression by binding to an operator sequence in the parDE promoter region. This DNA binding activity is crucial for the regulation of the relative abundance of toxin and antidote which ultimately determines life or death for the bacterial host and its daughter cells. In light scattering studies and gel filtration chromatography we observed the existence of a stable dimer of ParD in solution. The stoichiometry of ParD-DNA complex formation appeared to be 4:1, the molecular mass of the complex was 72.1 kDa. The alpha-helical content of ParD as determined by CD-spectrometry was 35%. The protein exhibited high thermostability with a T(M) of 64 degrees C and deltaH of 25 kcal/mol as shown by differential scanning calorimetry. Upon complex formation the T(M) increased by 10 degrees C. The thermal unfolding of the ParD protein was highly reversible as observed in repeated DSC scans of the same sample. The recovery of the native fold was proven by CD-spectroscopy.

Bacterial Proteins↗
PubMed1998

Control of cleavage site selection during mRNA 3' end formation by a yeast hnRNP.

Endonucleolytic cleavage of pre-mRNAs is the first step during eukaryotic mRNA 3' end formation. It has been proposed that cleavage factors CF IA, CF IB and CF II are required for pre-mRNA 3' end cleavage in yeast. CF IB is composed of a single polypeptide, Nab4p/Hrp1p, which is related to the A/B group of metazoan heterogeneous nuclear ribonucleoproteins (hnRNPs) that function as antagonistic regulators of 5' splice site selection. Here, we provide evidence that Nab4p/Hrp1p is not required for pre-mRNA 3' end endonucleolytic cleavage. We show that CF IA and CF II devoid of Nab4p/Hrp1p are sufficient to cleave a variety of RNA substrates but that cleavage occurs at multiple sites. Addition of Nab4p/Hrp1p prevents these alternative cleavages in a concentration-dependent manner, suggesting an essential and conserved role for some hnRNPs in pre-mRNA cleavage site selection.

Base Sequence↗
PubMed1998

Tad1p, a yeast tRNA-specific adenosine deaminase, is related to the mammalian pre-mRNA editing enzymes ADAR1 and ADAR2.

We have identified an RNA-specific adenosine deaminase (termed Tad1p/scADAT1) from Saccharomyces cerevisiae that selectively converts adenosine at position 37 of eukaryotic tRNAAla to inosine. The activity of purified recombinant Tad1p depends on the conformation of its tRNA substrate and the enzyme was found to be inactive on all other types of RNA tested. Mutant strains in which the TAD1 gene is disrupted are viable but lack Tad1p enzyme activity and their tRNAAla is not modified at position A37. Transformation of the mutant cells with the TAD1 gene restored enzyme activity. Tad1p has significant sequence similarity with the mammalian editing enzymes which act on specific precursor-mRNAs and on long double-stranded RNA. These findings suggest an evolutionary link between pre-mRNA editing and tRNA modification.

Adenosine Deaminase↗
PubMed1998

Coupling termination of transcription to messenger RNA maturation in yeast.

The direct association between messenger RNA (mRNA) 3'-end processing and the termination of transcription was established for the CYC1 gene of Saccharomyces cerevisiae. The mutation of factors involved in the initial cleavage of the primary transcript at the poly(A) site (RNA14, RNA15, and PCF11) disrupted transcription termination at the 3' end of the CYC1 gene. In contrast, the mutation of factors involved in the subsequent polyadenylation step (PAP1, FIP1, and YTH1) had little effect. Thus, cleavage factors link transcription termination of RNA polymerase II with pre-mRNA 3'-end processing.

Cytochrome c Group↗
PubMed1998

Human pre-mRNA cleavage factor Im is related to spliceosomal SR proteins and can be reconstituted in vitro from recombinant subunits.

Four polypeptides of 25, 59, 68, and 72 kDa copurify with the activity of human cleavage factor Im (CF Im) involved in pre-mRNA 3' end processing. We report here the cloning of the 25 and 68 kDa subunits and the reconstitution of functional CF Im25/68 from these two polypeptides. Several lines of evidence indicate that CF Im exists in at least two different forms. The 68 kDa polypeptide has a domain organization reminiscent of spliceosomal SR proteins. Analysis of the kinetics of the cleavage reaction indicates that interaction of CF Im with the RNA is one of the earliest steps in the assembly of the 3' end processing complex and facilitates the recruitment of other processing factors.

Amino Acid Sequence↗
PubMed1998

Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3'end formation of cellular pre-mRNAs.

Inhibition of the nuclear export of poly(A)-containing mRNAs caused by the influenza A virus NS1 protein requires its effector domain. Here, we demonstrate that the NS1 effector domain functionally interacts with the cellular 30 kDa subunit of CPSF, an essential component of the 3' end processing machinery of cellular pre-mRNAs. In influenza virus-infected cells, the NS1 protein is physically associated with CPSF 30 kDa. Binding of the NS1 protein to the 30 kDa protein in vitro prevents CPSF binding to the RNA substrate and inhibits 3' end cleavage and polyadenylation of host pre-mRNAs. The NS1 protein also inhibits 3' end processing in vivo, and the uncleaved pre-mRNA remains in the nucleus. Via this novel regulation of pre-mRNA 3' end processing, the NS1 protein selectively inhibits the nuclear export of cellular, and not viral, mRNAs.

Amino Acid Sequence↗
PubMed1998

Peri-implant microflora of implants with cemented and screw retained suprastructures.

The aims of this study were to compare clinical and microbiological features in the peri-implant area of implants carrying either screw retained or cemented suprastructures, and to investigate the relationship between the peri-implant microflora, the microbiota on the inner surface of removable suprastructures, and the periodontal microflora within the same subject. In 15 partially edentulous patients with ITI implants used as abutments for crown and bridge reconstructions, microbial samples were taken i) from the deepest periodontal pocket of each quadrant, ii) from the sulcus of the implants and iii) from the internal surface of the screw retained suprastructures. The samples were cultured using continuous anaerobic techniques. Five patients were found with both screw retained (S) and cemented (C) suprastructures. In these subjects the mean total cultivable counts were significantly higher in peri-implant samples from group C than in samples from group S. Furthermore, peri-implant samples of group S yielded a higher proportion of coccoid cells in the darkfield microscope and demonstrated absence of large spirochetes. From the 15 patients, Porphyromas gingivalis was detected in 10% of the periodontal samples and in only one peri-implant sample. Prevotella intermedia was detected in 33% of the periodontal and in 30% of the peri-implant samples. Fusobacterium spp. had a prevalence of 58% in the periodontal samples and was recovered from 50% of the peri-implant samples. Actinobacillus actinomycetemcomitans was not detected in any dental or peri-implant sample. In 1 case, however, the organism was recovered from the internal surface of the suprastructure. Linear regression analysis showed a significant relationship between the frequency of micro-organisms in peri-implant samples of group S and in samples from the inner surface of the suprastructure. Furthermore, there was a significant correlation between the incidence of micro-organisms in dental samples and i) in peri-implant samples of group S and ii) in samples from the internal suprastructure surface. These findings indicate, that the microbial leakage through the gap between the suprastructure and the abutment plays an important role in the bacterial colonization of the internal part of screw retained crowns and bridges. The study furthermore confirms the impact of the dental microflora on the microbial colonization of implants. This factor appears to be more important than the mode of fixation of the suprastructure.

Adult↗
PubMed1998

Effect of psychotherapy on the course of Crohn's disease. Results of the German prospective multicenter psychotherapy treatment study on Crohn's disease. German Study Group on Psychosocial Intervention in Crohn's Disease.

BACKGROUND: Our aim was to study the influence of psychotherapy in addition to a standardized corticosteroid treatment on the somatic and psychosocial course of Crohn's disease. METHODS: In a prospective, randomized multicenter study 108 of 488 patients received either drug treatment or, in the intervention group, additionally psychotherapy in the first half of the 2-year follow-up period. RESULTS: Eighty-four patients (77.8%) completed the somatic and 81 (75%) the psychosocial follow-up. Twenty-three per cent of the control group and 30% of the psychotherapy group showed episode-free courses; 29% and 17%, respectively, underwent surgery due to failure of drug treatment. The main analysis, which was based on subranking by number, duration, and severity of relapses, failed to show significant differences between the two groups (P = 0.125). The same result was obtained for the psychosocial status after 1 year in the main target criteria depression, anxiety, psychosocio-communicative status, and quality of life. CONCLUSIONS: The confirmatory analysis did not prove significantly better courses after additional psychotherapy. There was a tendency towards fewer operations.

Adolescent↗
PubMed1998

Tailing and 3'-end labeling of RNA with yeast poly(A) polymerase and various nucleotides.

We have tested conditions for the labeling and tailing the 3'-end of RNAs with yeast poly(A) polymerase. Conditions were optimized for addition of NTP, dNTP, or ddNTP nucleotides to RNA. ATP, GTP, and UTP were useful for adding homopolymer tracts of various lengths. The nonradioactive nucleotides biotin-N6-ATP and digoxigenin-11-UTP also were used efficiently.

Base Sequence↗
PubMed1997

RNA ligands selected by cleavage stimulation factor contain distinct sequence motifs that function as downstream elements in 3'-end processing of pre-mRNA.

Critical events in 3'-end processing of pre-mRNA are the recognition of the AAUAAA polyadenylation signal by cleavage and polyadenylation specificity factor (CPSF) and the binding of cleavage stimulation factor (CstF) via its 64-kDa subunit to the downstream element. The stability of this CPSF.CstF.RNA complex is thought to determine the efficiency of 3'-end processing. Since downstream elements reveal high sequence variability, in vitro selection experiments with highly purified CstF were performed to investigate the sequence requirements for CstF-RNA interaction. CstF was purified from calf thymus and from HeLa cells. Surprisingly, calf thymus CstF contained an additional, novel form of the 64-kDa subunit with a molecular mass of 70 kDa. RNA ligands selected by HeLa and calf thymus CstF contained three highly conserved sequence elements as follows: element 1 (AUGCGUUCCUCGUCC) and two closely related elements, element 2a (YGUGUYN0-4UUYAYUGYGU) and element 2b (UUGYUN0-4AUUUACU(U/G)N0-2YCU). All selected sequences tested functioned as downstream elements in 3'-end processing in vitro. A computer survey of the EMBL data library revealed significant homologies to all selected elements in naturally occurring 3'-untranslated regions. The majority of element 2a homologies was found downstream of coding sequences. Therefore, we postulate that this element represents a novel consensus sequence for downstream elements in 3'-end processing of pre-mRNA.

Animals↗
PubMed1997

A multisubunit 3' end processing factor from yeast containing poly(A) polymerase and homologues of the subunits of mammalian cleavage and polyadenylation specificity factor.

Polyadenylation is the second step in 3' end formation of most eukaryotic mRNAs. In Saccharomyces cerevisiae, this step requires three trans-acting factors: poly(A) polymerase (Pap1p), cleavage factor I (CF I) and polyadenylation factor I (PF I). Here, we describe the purification and subunit composition of a multiprotein complex containing Pap1p and PF I activities. PF I-Pap1p was purified to homogeneity by complementation of extracts mutant in the Fip1p subunit of PF I. In addition to Fip1p and Pap1p, the factor comprises homologues of all four subunits of mammalian cleavage and polyadenylation specificity factor (CPSF), as well as Ptalp, which previously has been implicated in pre-tRNA processing, and several as yet uncharacterized proteins. As expected for a PF I subunit, pta1-1 mutant extracts are deficient for polyadenylation in vitro. PF I also appears to be functionally related to CPSF, as it polyadenylates a substrate RNA more efficiently than Pap1p alone. Possibly, the observed interaction of the complex with RNA tethers Pap1p to its substrate.

Amino Acid Sequence↗