The Experts below are selected from a list of 270 Experts worldwide ranked by ideXlab platform
Norbert Polacek - One of the best experts on this subject based on the ideXlab platform.
-
the role of the universally conserved a2450 c2063 Base Pair in the ribosomal peptidyl transferase center
Nucleic Acids Research, 2010Co-Authors: Anna Chirkova, Marek Zywicki, Michaela Aigner, Matthias D Erlacher, Nina Clementi, Norbert PolacekAbstract:: Despite the fact that all 23S rRNA nucleotides that build the ribosomal peptidyl transferase ribozyme are universally conserved, standard and atomic mutagenesis studies revealed the nucleoBase identities being non-critical for catalysis. This indicates that these active site residues are highly conserved for functions distinct from catalysis. To gain insight into potential contributions, we have manipulated the nucleoBases via an atomic mutagenesis approach and have utilized these chemically engineered ribosomes for in vitro translation reactions. We show that most of the active site nucleoBases could be removed without significant effects on polypeptide production. Our data however highlight the functional importance of the universally conserved non-Watson-Crick Base Pair at position A2450-C2063. Modifications that disrupt this Base Pair markedly imPair translation activities, while having little effects on peptide bond formation, tRNA drop-off and ribosome-dependent EF-G GTPase activity. Thus it seems that disruption of the A2450-C2063 Pair inhibits a reaction following transpeptidation and EF-G action during the elongation cycle. Cumulatively our data are compatible with the hypothesis that the integrity of this A-C Wobble Base Pair is essential for effective tRNA translocation through the peptidyl transferase center during protein synthesis.
-
The role of the universally conserved A2450–C2063 Base Pair in the ribosomal peptidyl transferase center
Nucleic Acids Research, 2010Co-Authors: Anna Chirkova, Marek Zywicki, Michaela Aigner, Matthias D Erlacher, Nina Clementi, Norbert PolacekAbstract:: Despite the fact that all 23S rRNA nucleotides that build the ribosomal peptidyl transferase ribozyme are universally conserved, standard and atomic mutagenesis studies revealed the nucleoBase identities being non-critical for catalysis. This indicates that these active site residues are highly conserved for functions distinct from catalysis. To gain insight into potential contributions, we have manipulated the nucleoBases via an atomic mutagenesis approach and have utilized these chemically engineered ribosomes for in vitro translation reactions. We show that most of the active site nucleoBases could be removed without significant effects on polypeptide production. Our data however highlight the functional importance of the universally conserved non-Watson-Crick Base Pair at position A2450-C2063. Modifications that disrupt this Base Pair markedly imPair translation activities, while having little effects on peptide bond formation, tRNA drop-off and ribosome-dependent EF-G GTPase activity. Thus it seems that disruption of the A2450-C2063 Pair inhibits a reaction following transpeptidation and EF-G action during the elongation cycle. Cumulatively our data are compatible with the hypothesis that the integrity of this A-C Wobble Base Pair is essential for effective tRNA translocation through the peptidyl transferase center during protein synthesis.
Anna Chirkova - One of the best experts on this subject based on the ideXlab platform.
-
the role of the universally conserved a2450 c2063 Base Pair in the ribosomal peptidyl transferase center
Nucleic Acids Research, 2010Co-Authors: Anna Chirkova, Marek Zywicki, Michaela Aigner, Matthias D Erlacher, Nina Clementi, Norbert PolacekAbstract:: Despite the fact that all 23S rRNA nucleotides that build the ribosomal peptidyl transferase ribozyme are universally conserved, standard and atomic mutagenesis studies revealed the nucleoBase identities being non-critical for catalysis. This indicates that these active site residues are highly conserved for functions distinct from catalysis. To gain insight into potential contributions, we have manipulated the nucleoBases via an atomic mutagenesis approach and have utilized these chemically engineered ribosomes for in vitro translation reactions. We show that most of the active site nucleoBases could be removed without significant effects on polypeptide production. Our data however highlight the functional importance of the universally conserved non-Watson-Crick Base Pair at position A2450-C2063. Modifications that disrupt this Base Pair markedly imPair translation activities, while having little effects on peptide bond formation, tRNA drop-off and ribosome-dependent EF-G GTPase activity. Thus it seems that disruption of the A2450-C2063 Pair inhibits a reaction following transpeptidation and EF-G action during the elongation cycle. Cumulatively our data are compatible with the hypothesis that the integrity of this A-C Wobble Base Pair is essential for effective tRNA translocation through the peptidyl transferase center during protein synthesis.
-
The role of the universally conserved A2450–C2063 Base Pair in the ribosomal peptidyl transferase center
Nucleic Acids Research, 2010Co-Authors: Anna Chirkova, Marek Zywicki, Michaela Aigner, Matthias D Erlacher, Nina Clementi, Norbert PolacekAbstract:: Despite the fact that all 23S rRNA nucleotides that build the ribosomal peptidyl transferase ribozyme are universally conserved, standard and atomic mutagenesis studies revealed the nucleoBase identities being non-critical for catalysis. This indicates that these active site residues are highly conserved for functions distinct from catalysis. To gain insight into potential contributions, we have manipulated the nucleoBases via an atomic mutagenesis approach and have utilized these chemically engineered ribosomes for in vitro translation reactions. We show that most of the active site nucleoBases could be removed without significant effects on polypeptide production. Our data however highlight the functional importance of the universally conserved non-Watson-Crick Base Pair at position A2450-C2063. Modifications that disrupt this Base Pair markedly imPair translation activities, while having little effects on peptide bond formation, tRNA drop-off and ribosome-dependent EF-G GTPase activity. Thus it seems that disruption of the A2450-C2063 Pair inhibits a reaction following transpeptidation and EF-G action during the elongation cycle. Cumulatively our data are compatible with the hypothesis that the integrity of this A-C Wobble Base Pair is essential for effective tRNA translocation through the peptidyl transferase center during protein synthesis.
En-duo Wang - One of the best experts on this subject based on the ideXlab platform.
-
the g3 u70 independent trna recognition by human mitochondrial alanyl trna synthetase
Nucleic Acids Research, 2019Co-Authors: Qiyu Zeng, En-duo Wang, Guixin Peng, Guang Li, Jingbo Zhou, Wenqiang Zheng, Meiqin Xue, Xiaolong ZhouAbstract:Alanyl-tRNA synthetases (AlaRSs) from three domains of life predominantly rely on a single Wobble Base Pair, G3-U70, of tRNA(Ala) as a major determinant. However, this Base Pair is divergent in human mitochondrial tRNA(Ala), but instead with a translocated G5-U68. How human mitochondrial AlaRS (hmtAlaRS) recognizes tRNA(Ala), in particular, in the acceptor stem region, remains unknown. In the present study, we found that hmtAlaRS is a monomer and recognizes mitochondrial tRNA(Ala) in a G3-U70-independent manner, requiring several elements in the acceptor stem. In addition, we found that hmtAlaRS misactivates noncognate Gly and catalyzes strong transfer RNA (tRNA)-independent pre-transfer editing for Gly. A completely conserved residue outside of the editing active site, Arg(663), likely functions as a tRNA translocation determinant to facilitate tRNA entry into the editing domain during editing. Finally, we investigated the effects of the severe infantile-onset cardiomyopathy-associated R592W mutation of hmtAlaRS on the canonical enzymatic activities of hmtAlaRS. Overall, our results provide fundamental information about tRNA recognition and deepen our understanding of translational quality control mechanisms by hmtAlaRS.
-
Discrimination of tRNA Leu Isoacceptors by the Mutants of Escherichia coli Leucyl-tRNA Synthetase in Editing †
Biochemistry, 2002Co-Authors: En-duo WangAbstract:Leucyl-tRNA synthetase (LeuRS), one of the class la aminoacyl-tRNA synthetases, joins Leu to tRNA(Leu) and excludes noncognate amino acids in protein synthesis. In this study, Escherichia coli LeuRS mutants at amino acid E292, which was located in the connective polypeptide I insertion region, were synthesized. Although mutated LeuRS showed little change in structure compared with wild-type LeuRS, the mutants were imPaired in activity to varying extents. It was also showed that mutations did not affect the adenylation reaction. However, mutated LeuRS can mischarge tRNA(Leu) isoacceptors tRNA(1)(Leu) or tRNA(2)(Leu) with isoleucine to different extents. Isoleucylation of tRNA(1)(Leu) was more than that of tRNA(2)(Leu). The mutant LeuRS-E292S, which was picked out as an example for the investigation of the relationship between tRNALeu isoacceptors and editing function, can discriminate the Watson-Crick Base Pair of the first Base Pair of tRNA(Leu) from the Wobble Base Pair. The tRNA(Leu) with the Watson-Crick Base Pair may result in more isoleucylated product than that with the Wobble Base Pair. The same phenomenon happened to another mutant, LeuRS-A293D. It seems that the flexibility of the first Base Pair affects the editing reaction of LeuRS. The results indicate that the flexibility of the first Base Pair of tRNALeu may probably affect the mischarged 3'-end of tRNA(Leu) shuttling from synthetic site to editing site and that the transferred acceptor arm of tRNA(Leu) may interact with LeuRS in the region around E292.
Nassim Usman - One of the best experts on this subject based on the ideXlab platform.
-
The 2,6-diaminopurine riboside.5-methylisocytidine Wobble Base Pair: an isoenergetic substitution for the study of G.U Pairs in RNA.
Biochemistry, 1994Co-Authors: Scott A. Strobel, Thomas R. Cech, Nassim Usman, Leonid BeigelmanAbstract:Phylogenetically invariant G.U Wobble Pairs are present in a wide variety of RNA's. As a means to study the contribution of individual chemical groups within a G.U Pair, we have synthesized and thermodynamically characterized oligoribonucleotides containing the unnatural nucleosides 2,6-diaminopurine riboside (DAP) and 5-methylisocytidine (MeiC). The DAP.MeiC Pair at the end of an RNA duplex is as stable as a G.U Pair, consistent with formation of a Wobble Base Pair with two hydrogen bonds. DAP.MeiC is a valuable substitution for the study of G.U Wobble Pairs because it is conformationally similar to the G.U Pair, but has a different array of functional groups in the major and minor grooves of the duplex and a reversed hydrogen bonding polarity between the Bases. We also report the stability of several other terminal Pairs proposed to be in a Wobble configuration including inosine.U (I.U), A.MeiC, DAP.C, A.C, G.5-methyl-U,2'-deoxyguanosine.U, and 2'-deoxy-7-deazaguanosine.U. These Pairs present a diversity of functional group substitutions in the context of a Wobble conformation. Comparison of Wobble Pairs with and without the N2 exocyclic amine, i.e., G.U vs I.U, DAP.MeiC vs A.MeiC, and DAP.C vs A.C, demonstrates that the amine does not contribute to Base Pairing stability when the Pair is located at the terminal position of the RNA duplex. However, at a position internal to the duplex, the exocyclic amine does improve helix stability. An internal I.U Pair is less stable (approximately 1 kcal.mol-1) than an internal G.U Pair, and substantially less stable (approximately 2 kcal.mol-1) than an internal A-U Pair. These data provide quantitation for the reduced duplex stability observed upon conversion of A-U to I.U Pairs by double-stranded RNA adenosine deaminase (dsRAD). This collection of Wobble Pairs will help identify the contribution made by individual functional groups in RNA/protein interactions and in the tertiary folding of RNA.
-
synthesis of rna containing inosine analysis of the sequence requirements for the 5 splice site of the tetrahymena group i intron
Nucleic Acids Research, 1991Co-Authors: Rachel Green, Jack W Szostak, Steven A Benner, Alexander Rich, Nassim UsmanAbstract:Two protected derivatives of the ribonucleoside inosine have been prepared to serve as building blocks for phosphoramidite-Based synthesis of RNA. Two different synthetic routes address the unusual solubility characteristics of inosine and its derivatives. The final products of the different synthetic pathways, 5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsiyl) inosine 3'-O-(beta-cyanoethyldiisopropylamino) phosphoramidite 5a, and O6-p-nitrophenylethyl-5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsilyl) inosine 3'-O-(methyldiisopropylamino) phosphoramidite 5b, were chemically incorporated into short oligoribonucleotides which also contained the four standard ribonucleoside Bases. The oligomers were chosen to study Base-specific interactions between an RNA substrate and an RNA enzyme derived from the Group I Tetrahymena self-splicing intron. The oligomers were shown to be biochemically competent using a trans cleavage assay with the modified Tetrahymena intron. The results confirm the dependence of the catalytic activity on a Wobble Base Pair, rather than a Watson-Crick Base Pair, in the helix at the 5'-splice site. Furthermore, comparison of guanosine and inosine in a Wobble Base Pair allows one to assess the importance of the guanine 2-amino group for biological activity. The preparation of the inosine phosphoramidites adds to the repertoire of Base analogues available for the study of RNA catalysis and RNA-protein interactions.
-
Synthesis of RNA containing inosine: analysis of the sequence requirements for the 5′ splice site of the Tetrahymena group I intron
Nucleic acids research, 1991Co-Authors: Rachel Green, Jack W Szostak, Steven A Benner, Alexander Rich, Nassim UsmanAbstract:Two protected derivatives of the ribonucleoside inosine have been prepared to serve as building blocks for phosphoramidite-Based synthesis of RNA. Two different synthetic routes address the unusual solubility characteristics of inosine and its derivatives. The final products of the different synthetic pathways, 5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsiyl) inosine 3'-O-(beta-cyanoethyldiisopropylamino) phosphoramidite 5a, and O6-p-nitrophenylethyl-5'-O-(dimethoxytrityl)-2'-O-(t-butyldimethylsilyl) inosine 3'-O-(methyldiisopropylamino) phosphoramidite 5b, were chemically incorporated into short oligoribonucleotides which also contained the four standard ribonucleoside Bases. The oligomers were chosen to study Base-specific interactions between an RNA substrate and an RNA enzyme derived from the Group I Tetrahymena self-splicing intron. The oligomers were shown to be biochemically competent using a trans cleavage assay with the modified Tetrahymena intron. The results confirm the dependence of the catalytic activity on a Wobble Base Pair, rather than a Watson-Crick Base Pair, in the helix at the 5'-splice site. Furthermore, comparison of guanosine and inosine in a Wobble Base Pair allows one to assess the importance of the guanine 2-amino group for biological activity. The preparation of the inosine phosphoramidites adds to the repertoire of Base analogues available for the study of RNA catalysis and RNA-protein interactions.
Max Grossenbacher - One of the best experts on this subject based on the ideXlab platform.
-
2-Aminopurine is a Reporter for the Formation of the C·A+ Wobble Base Pair in U6 snRNA
The FASEB Journal, 2016Co-Authors: Max GrossenbacherAbstract:The spliceosome is a multimegadalton complex that is found in all eukaryotes and is involved in the excision of introns and ligation of exons to create mature RNA from pre-mRNA. The U6 subunit of the spliceosome contains a highly conserved internal-stem loop (ISL), which is essential to the activation and catalytic function of the spliceosome. The ISL has a 1X2 bulge that consists of a C67 ·A79+ Wobble Base Pair (pKa 6.5) and an unPaired U80 nucleotide. The formation of C67· A79+ results in the Base flipping of U80 out of the RNA helix, thus, stabilizing the formation of a continuous A-form helix. At a pH above 6.5, U80 is stacked within the RNA helix, resulting in a bend in the helix that favors ion interactions. We examined the structural dynamics of U6 RNA using 2-aminopurine (2-AP), a fluorescent analog of adenine and guanine, that was substituted into U6 RNA constructs for A78. Thermal denaturation and fluorescent studies were used to measure the effects of pH and Mg2+ on the stability of U6 wild typ...
-
2 aminopurine is a reporter for the formation of the c a Wobble Base Pair in u6 snrna
The FASEB Journal, 2016Co-Authors: Max GrossenbacherAbstract:The spliceosome is a multimegadalton complex that is found in all eukaryotes and is involved in the excision of introns and ligation of exons to create mature RNA from pre-mRNA. The U6 subunit of the spliceosome contains a highly conserved internal-stem loop (ISL), which is essential to the activation and catalytic function of the spliceosome. The ISL has a 1X2 bulge that consists of a C67 ·A79+ Wobble Base Pair (pKa 6.5) and an unPaired U80 nucleotide. The formation of C67· A79+ results in the Base flipping of U80 out of the RNA helix, thus, stabilizing the formation of a continuous A-form helix. At a pH above 6.5, U80 is stacked within the RNA helix, resulting in a bend in the helix that favors ion interactions. We examined the structural dynamics of U6 RNA using 2-aminopurine (2-AP), a fluorescent analog of adenine and guanine, that was substituted into U6 RNA constructs for A78. Thermal denaturation and fluorescent studies were used to measure the effects of pH and Mg2+ on the stability of U6 wild typ...
