scholarly journals Loss of a Universal tRNA Feature

2006 ◽  
Vol 189 (5) ◽  
pp. 1954-1962 ◽  
Author(s):  
Chunxia Wang ◽  
Bruno W. Sobral ◽  
Kelly P. Williams
Keyword(s):  
Sinorhizobium Meliloti ◽  
Caulobacter Crescentus ◽  
Trna Synthetase ◽  
Binding Domain ◽  
Charging System ◽  
A Site ◽  
Extra Nucleotide

ABSTRACT tRNAHis has thus far always been found with one of the most distinctive of tRNA features, an extra 5′ nucleotide that is usually a guanylate. tRNAHis genes in a disjoint alphaproteobacterial group comprising the Rhizobiales, Rhodobacterales, Caulobacterales, Parvularculales, and Pelagibacter generally fail to encode this extra guanylate, unlike those of other alphaproteobacteria and bacteria in general. Rather than adding an extra 5′ guanylate posttranscriptionally as eukaryotes do, evidence is presented here that two of these species, Sinorhizobium meliloti and Caulobacter crescentus, simply lack any extra nucleotide on tRNAHis. This loss correlates with changes at the 3′ end sequence of tRNAHis and at many sites in histidyl-tRNA synthetase that might be expected to affect tRNAHis recognition, in the flipping loop, the insertion domain, the anticodon-binding domain, and the motif 2 loop. The altered tRNA charging system may have affected other tRNA charging systems in these bacteria; for example, a site in tRNAGlu sequences was found to covary with tRNAHis among alphaproteobacteria.

scholarly journals The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication ofVibrio choleraeChromosome 2

mBio ◽  
2017 ◽  
Vol 8 (2) ◽  
Author(s):  
Jyoti K. Jha ◽  
Mi Li ◽  
Rodolfo Ghirlando ◽  
Lisa M. Miller Jenkins ◽  
Alexander Wlodawer ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Molecular Chaperone ◽  
Binding Domain ◽  
Chromosome 2 ◽  
Dynamic Nature ◽  
Dimerization Domain ◽  
Inhibitory Mechanisms ◽  
Chaperone Dnak ◽  
Suppression Mechanism ◽  
A Site

ABSTRACTReplication ofVibrio choleraechromosome 2 (Chr2) depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB) to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding. We found that mutations inrctBthat reduce DnaK binding also reduce 12-mer binding and initiation. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally. Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. One suppressing change was in a dimerization domain which is folded similarly to the initiator of an iteron plasmid—the presumed progenitor of Chr2. In plasmids, DnaK promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of RctB and inactivated it, indicating an alternative suppression mechanism. Paradoxically, although DnaK increases 39-mer binding, the increase was also achieved by inactivating the DnaK binding site of RctB. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition) when prevented from binding DnaK. Taken together, our results reveal an important feature of the transition from plasmid to chromosome: the Chr2 initiator retains the plasmid-like dimerization domain and its control by chaperones but uses the chaperones in an unprecedented way to control the inhibitory 39-mer binding.IMPORTANCEThe capacity of proteins to undergo remodeling provides opportunities to control their function. However, remodeling remains a poorly understood aspect of the structure-function paradigm due to its dynamic nature. Here we have studied remodeling of the initiator of replication ofVibrio choleraeChr2 by the molecular chaperone, DnaK. We show that DnaK binds to a site on the Chr2 initiator (RctB) that promotes initiation by reducing the initiator’s propensity to dimerize. Dimerization of the initiator of the putative plasmid progenitor of Chr2 is also reduced by DnaK, which promotes initiation. Paradoxically, the DnaK binding also promotes replication inhibition by reducing an autoinhibitory activity of RctB. In the plasmid-to-chromosome transition, it appears that the initiator has acquired an autoinhibitory activity and along with it a new chaperone activity that apparently helps to control replication inhibition independently of replication promotion.

scholarly journals High-resolution crystal structures of alternate forms of the human CD44 hyaluronan-binding domain reveal a site for protein interaction

2014 ◽  
Vol 70 (9) ◽  
pp. 1155-1161 ◽  
Author(s):  
Li-Kai Liu ◽  
Barry Finzel
Keyword(s):  
High Resolution ◽  
Crystal Structures ◽  
Crystal Form ◽  
Binding Domain ◽  
Structural Deformation ◽  
Monoclinic Form ◽  
Alternate Forms ◽  
Binding Groove ◽  
A Site ◽  
Hyaluronan Binding

Two new crystal structures of the extracellular hyaluronan-binding domain of human CD44 are described at high resolution. A hexagonal crystal form at 1.60 Å resolution and a monoclinic form at 1.08 Å resolution both have two molecules in the asymmetric unit arranged about a similar noncrystallographic twofold axis of symmetry. These structures are compared with those previously reported at 2.20 Å resolution to show that the fold is quite resistant to structural deformation in different crystal environments. Unexpectedly, a short peptide is found in the monoclinic crystals at a site remote from the known hyaluronan-binding groove. The peptide with a valine at the carboxy-terminus must have co-purified from the bacterial expression host and binds on the opposite side of the domain from the known hyaluronan-binding groove. This opportunistic binding may identify a site of interaction used as CD44 assembles with other proteins to accomplish effective signaling regarding changes to the extracellular environment.

scholarly journals Suppression of Amber Codons in Caulobacter crescentus by the Orthogonal Escherichia coli Histidyl-tRNA Synthetase/tRNAHis Pair

PLoS ONE ◽  
2013 ◽  
Vol 8 (12) ◽  
pp. e83630 ◽  
Author(s):  
Jae-hyeong Ko ◽  
Paula Montero Llopis ◽  
Jennifer Heinritz ◽  
Christine Jacobs-Wagner ◽  
Dieter Söll
Keyword(s):  
Escherichia Coli ◽  
Caulobacter Crescentus ◽  
Trna Synthetase

Optimization of the posttranslational click modification of proteins

2011 ◽  
Vol 76 (9) ◽  
pp. 1089-1101
Author(s):  
Milan Vrabel ◽  
Emine Kaya ◽  
Stefan Prill ◽  
Veronika Ehmke ◽  
Thomas Carell
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Fluorescent Protein ◽  
Click Reaction ◽  
Yellow Fluorescent Protein ◽  
Trna Synthetase ◽  
Site Specific ◽  
Specific Manner ◽  
Modified Proteins ◽  
A Site

In order to develop efficient methods that would enable the synthesis of posttranslationaly modified proteins in a site-specific manner we have adopted the orthogonal pyrrolysyl-tRNA synthetase/tRNA pair to genetically encode various pyrrolysine analogs, which we were able to insert into the yellow fluorescent protein (YFP). These experiments showed that the alkene and alkyne containing amino acids 5 and 6 are superior substrates for the pyrrolysyl-tRNA synthetase and that they can be successfully incorporated into proteins. Using the Cu(I)-catalyzed Huisgen–Meldal–Sharpless click reaction, the alkyne containing YFP was finally glycosylated with various sugars. We confirmed the presence of the modified amino acids as well as the corresponding sugar modifications by HPLC-MS/MS mass spectrometry.

scholarly journals Subinhibitory Concentrations of Bacteriostatic Antibiotics Induce relA-Dependent and relA-Independent Tolerance to β-Lactams

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Pavel Kudrin ◽  
Vallo Varik ◽  
Sofia Raquel Alves Oliveira ◽  
Jelena Beljantseva ◽  
Teresa Del Peso Santos ◽  
...  
Keyword(s):  
Trna Synthetase ◽  
Growth Stress ◽  
Amino Acid Starvation ◽  
Bacterial Metabolism ◽  
Persister Cells ◽  
Biochemical System ◽  
Content Type ◽  
E Coli ◽  
Bacterial Cultures ◽  
A Site

ABSTRACTThe nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence. During amino acid starvation, theEscherichia coli(p)ppGpp synthetase RelA is activated by deacylated tRNA in the ribosomal A-site. An increase in (p)ppGpp is believed to drive the formation of antibiotic-tolerant persister cells, prompting the development of strategies to inhibit (p)ppGpp synthesis. We show that in a biochemical system from purifiedE. colicomponents, the antibiotic thiostrepton efficiently inhibits RelA activation by the A-site tRNA. In bacterial cultures, the ribosomal inhibitors thiostrepton, chloramphenicol, and tetracycline all efficiently abolish accumulation of (p)ppGpp induced by the Ile-tRNA synthetase inhibitor mupirocin. This abolishment, however, does not reduce the persister level. In contrast, the combination of dihydrofolate reductase inhibitor trimethoprim with mupirocin, tetracycline, or chloramphenicol leads to ampicillin tolerance. The effect is independent of RelA functionality, specific to β-lactams, and not observed with the fluoroquinolone norfloxacin. These results refine our understanding of (p)ppGpp's role in antibiotic tolerance and persistence and demonstrate unexpected drug interactions that lead to tolerance to bactericidal antibiotics.

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