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PubMed1997

Influence of genomic G+C content on average amino-acid composition of proteins from 59 bacterial species.

The amino-acid composition of 23,490 proteins from 59 bacterial species was analyzed as a function of genomic G+C content. Observed amino-acid frequencies were compared with those expected from a neutral model assuming the absence of selection on average protein composition. Integral membrane proteins and non-integral membrane proteins were analyzed separately. The average deviation from this neutral model shows that there is a selective pressure increasing content in charged amino acids for non-integral membrane proteins, and content in hydrophobic amino acids for integral membrane proteins. Amino-acid frequencies were greatly influenced by genomic G+C content, but the influence was found to be often weaker than predicted. This may be evidence for a selective pressure, maintaining most amino-acid frequencies close to an optimal value. Concordance between the genetic code and protein composition is discussed in the light of this observation.

Amino Acids↗
PubMed2000

Expression and antigenic characterization of recombinant Mycoplasma agalactiae P48 major surface protein.

The gene encoding the P48 major surface lipoprotein of M. agalactiae has been recently characterised. Since its product plays an important role in the immune response of infected animals, in this study we analysed a recombinant P48 expressed in E. coli. Multiple point mutations were introduced by site directed mutagenesis in order to convert four tryptophan TGA codons, which are a typical feature of the mycoplasma genetic code, into the standard TGG. The mutated p48 gene was subcloned into pGex-2T and expressed in fusion with glutathione-S transferase. Following purification steps, P48 was eluted from carrier protein by thrombin digestion and used in Western blot and indirect ELISA using well-characterised sheep sera. Results demonstrate that specific antibodies against P48 are detected 3 weeks after onset of clinical disease and the recombinant P48 is a diagnostically relevant marker of M. agalactiae infection.

Animals↗
PubMedDate not supplied

Retrovirus producer cells encoding antisense VEGF prolong survival of rats with intracranial GS9L gliomas.

With increasing size tumors are continually dependent on a functional blood vessel system to guarantee the supply with oxygen and nutrients. Vascular endothelial growth factor (VEGF) is a key mediator not only of developmental but also of hypoxia-mediated and tumor-induced angiogenesis. Gene therapy using antisense VEGF with the aim to inhibit tumor angiogenesis may be a successful strategy for the treatment of highly vascular and invasive malignant gliomas. We investigated whether retrovirus producer cells encoding antisense VEGF can be used for in vivo gene transfer. The full length mouse VEGF164 cDNA was cloned in a sense and antisense direction into the retroviral expression vector pLEN. pLEN-VEGF (sense) and pLEN-FGEV (antisense) expression vectors were used to transfect the packaging cell line GP + E86 and to establish ecotropic virus producer cell lines. GP + E86:LEN-FGEV (#5) cells showed high expression of antisense VEGF mRNA, whereas GP+ E86:LEN-VEGF (#8) showed high expression of sense VEGF mRNA and active VEGF protein. Co-implantation of GS-9L cells with retrovirus producing cells containing the antisense VEGF construct into the brains of syngeneic rats showed a statistically significant inhibition of tumor growth and prolongation of survival time, while co-implantation of retrovirus producer cells containing the sense VEGF expression vector resulted in an increasing tumor growth and reduced survival time of the rats compared to control animals. Histological analysis of the tumors co-implanted with GP + E86:LEN-FGEV (#5) cells showed the suppression of angiogenesis, high degree of necrosis and no evidence of a significant immune response. Expression of antisense VEGF mRNA in these tumors was confirmed by in situ hybridization analysis. This is the first report demonstrating the potential utility of virus producer cells as in vivo gene transfer vehicles for antisense VEGF gene therapy of malignant gliomas.

Animals↗
PubMed1998

Recent advances in producing and selecting functional proteins by using cell-free translation.

Prokaryotic and eukaryotic in vitro translation systems have recently become the focus of increasing interest for tackling fundamental problems in biochemistry. Cell-free systems can now be used to study the in vitro assembly of membrane proteins and viral particles, rapidly produce and analyze protein mutants, and enlarge the genetic code by incorporating unnatural amino acids. Using in vitro translation systems, display techniques of great potential have been developed for protein selection and evolution. Furthermore, progress has been made to efficiently produce proteins in batch or continuous cell-free translation systems and to elucidate the molecular causes of low yield and find possible solutions for this problem.

Animals↗
PubMed2001

Active site of an aminoacyl-tRNA synthetase dissected by energy-transfer-dependent fluorescence.

Aminoacyl-tRNA synthetases establish the rules of the genetic code by catalyzing attachment of amino acids to specific transfer RNAs (tRNAs) that bear the anticodon triplets of the code. Each of the 20 amino acids has its own distinct aminoacyl-tRNA synthetase. Here we use energy-transfer-dependent fluorescence from the nucleotide probe N-methylanthraniloyl dATP (mdATP) to investigate the active site of a specific aminoacyl-tRNA synthetase. Interaction of the enzyme with the cognate amino acid and formation of the aminoacyl adenylate intermediate were detected. In addition to providing a convenient tool to characterize enzymatic parameters, the probe allowed investigation of the role of conserved residues within the active site. Specifically, a residue that is critical for binding could be distinguished from one that is important for the transition state of adenylate formation. Amino acid binding and adenylate synthesis by two other aminoacyl-tRNA synthetases was also investigated with mdATP. Thus, a key step in the synthesis of aminoacyl-tRNA can in general be dissected with this probe.

Amino Acyl-tRNA Synthetases↗
PubMed2002

Identification of novel antifungal nonapeptides through the screening of combinatorial peptide libraries based on a hexapeptide motif.

Four sets of mixture based nonapeptide libraries derived from an antifungal hexapeptide pharmacophore Arg-D-Trp-D-Phe-Ile-D-Phe-His-NH(2) (II) have been synthesized. The three C-terminal positions 7, 8 and 9 were subject to randomization using 19 genetically coded amino acids. They were then screened for their antifungal activity against Candida albicans and Cryptococcus neoformans in order to quantify inhibition at each step of the nonapeptide sublibrary deconvolution. The studies led to the identification of several novel nonapeptides with potent antifungal activity. Two of the nonapeptides exhibited approximately 17-fold increase in the activity in comparison to the lead hexapeptide motif His-D-Trp-D-Phe-Phe-D-Phe-Lys-NH(2) (I) against C. albicans.

Amino Acid Motifs↗
PubMed2002

Ribosome structure: revisiting the connection between translational accuracy and unconventional decoding.

The ribosome is a molecular machine that converts genetic information in the form of RNA, into protein. Recent structural studies reveal a complex set of interactions between the ribosome and its ligands, mRNA and tRNA, that indicate ways in which the ribosome could avoid costly translational errors. Ribosomes must decode each successive codon accurately, and structural data provide a clear indication of how ribosomes limit recruitment of the wrong tRNA (sense errors). In a triplet-based genetic code there are three potential forward reading frames, only one of which encodes the correct protein. Errors in which the ribosome reads a codon out of the normal reading frame (frameshift errors) occur less frequently than sense errors, although it is not clear from structural data how these errors are avoided. Some mRNA sequences, termed programmed-frameshift sites, cause the ribosome to change reading frame. Based on recent work on these sites, this article proposes that the ribosome uses the structure of the codon-anticodon complex formed by the peptidyl-tRNA, especially its wobble interaction, to constrain the incoming aminoacyl-tRNA to the correct reading frame.

Protein Biosynthesis↗
PubMed1997

A strategy of tRNA recognition that includes determinants of RNA structure.

Recognition of tRNAs by aminoacyl tRNA synthetases establishes the connection between amino acids and anticodon triplets of the genetic code. Although anticodons and nucleotides adjacent to the amino acid attachment site are generally important, the tertiary structural framework of tRNAs has recently been implicated to have a role in tRNA recognition. A G15:G48 tertiary hydrogen base pair of E. coli tRNA(Cys) is important for recognition of the tRNA by cysteine tRNA synthetase. This base pair is proposed to consist of N2:N3, rather than N1:O6, hydrogen bonds. The reproduction of the hydrogen pairing scheme of tRNA(Gly). This reproduction required an A13:A22 mismatch in the dihyrouridine stem. To determine if A13:A22 is a determinant of the structural features of G15:G48, we investigated the A15:U48 and A15:A48 variants of tRNA(Gly) which harbored specific substitutions of A13:A22. We show here that introduction of A13:A22 to both tRNA frameworks confers structural features similar to those of G15:G48 in E. coli tRNA(Cys). These structural features are accompanied by efficient recognition of both tRNAs by cysteine tRNA synthetase. Substitution of A13:A22 with U13:A22 alters the structural features at 15:48 and impairs tRNA recognition. The dependence on A13:22 for tRNA recognition has a distinct similarity to that of E. coli tRNA(Cys) and to that of the G15:G48 variant of tRNA(Gly). The results have implications for the design and manipulation of RNA structural elements as the basis for tRNA recognition.

Acetylation↗
PubMed2003

Comparison of X-ray crystal structure of the 30S subunit-antibiotic complex with NMR structure of decoding site oligonucleotide-paromomycin complex.

Aminoglycoside antibiotics that bind to 16S ribosomal RNA in the aminoacyl-tRNA site (A site) cause misreading of the genetic code and inhibit translocation. Structures of an A site RNA oligonucleotide free in solution and bound to the aminoglycosides paromomycin or gentamicin C1a have been determined by NMR. Recently, the X-ray crystal structure of the entire 30S subunit has been determined, free and bound to paromomycin. Distinct differences were observed in the crystal structure, particularly at A1493. Here, the NMR structure of the oligonucleotide-paromomycin complex was determined with higher precision and is compared with the X-ray crystal structure of the 30S subunit complex. The comparison shows the validity of both structures in identifying critical interactions that affect ribosome function.

Anti-Bacterial Agents↗
PubMed1997

The crystal structure of phenylalanyl-tRNA synthetase from thermus thermophilus complexed with cognate tRNAPhe.

BACKGROUND: In the translation of the genetic code each aminoacyl-tRNA synthetase (aaRS) must recognize its own (cognate) tRNA and attach the corresponding amino acid to the acceptor end of tRNA, discriminating all the others. The(alphabeta)2 phenylalanyl-tRNA synthetase (PheRS) is one of the most complex enzymes in the aaRS family and is characterized by anomalous charging properties. Structurally, the enzyme belongs to class II aaRSs, as its catalytic domain is built around an antiparallel beta sheet, but functionally it resembles class I as it aminoacylates the 2'OH of the terminal ribose of tRNA (class II aaRSs aminoacylate the 3'OH). With the availability of the three-dimensional structure of the complex between multisubunit PheRS and tRNAPhe, a fuller picture of the specific tRNA-aaRS interactions is beginning to emerge. RESULTS: The crystal structure of Thermus thermophilus PheRS complexed with cognate tRNA has been solved at 3.28 A resolution. It reveals that one tRNAPhe molecule binds across all four PheRS subunits. The interactions of PheRS with tRNA stabilize the flexible N-terminal part of the alpha subunit, which appeared to form the enzyme's 11th domain, comprising a coiled-coil structure (helical arm) built up of two long antiparallel alpha helices. The helical arms are similar to those observed in SerRS and are in the same relative orientation with respect to the catalytic domain. Anticodon recognition upon tRNA binding is performed by the B8 domain, the structure of which is similar to that of the RNA-binding domain (RBD) of the small spliceosomal protein U1A. The Th. thermophilus PheRS approaches the anticodon loop from the minor groove side. CONCLUSIONS: The mode of interactions with tRNA explains the absolute necessity for the (alphabeta)2 architecture of PheRS. The interactions of tRNAPhe with PheRS and particularly with the coiled-coil domain of the alpha subunit result in conformational changes in TPsiC and D loops seen by comparison with uncomplexed yeast tRNAPhe. The tRNAPhe is a newly recognized type of RNA molecule specifically interacting with the RBD fold. In addition, a new type of anticodon-binding domain emerges in the aaRS family. The uniqueness of PheRS in charging 2'OH of tRNA is dictated by the size of its adenine-binding pocket and by the local conformation of the tRNA's CCA end.

Amino Acyl-tRNA Synthetases↗
PubMed1998

tRNA(Pro) anticodon recognition by Thermus thermophilus prolyl-tRNA synthetase.

BACKGROUND: Most aminoacyl-tRNA synthetases (aaRSs) specifically recognize all or part of the anticodon triplet of nucleotides of their cognate tRNAs. Class IIa and class IIb aaRSs possess structurally distinct tRNA anticodon-binding domains. The class IIb enzymes (LysRS, AspRS and AsnRS) have an N-terminal beta-barrel domain (OB-fold); the interactions of this domain with the anticodon stem-loop are structurally well characterised for AspRS and LysRS. Four out of five class IIa enzymes (ProRS, ThrRS, HisRS and GlyRS, but not SerRS) have a C-terminal anticodon-binding domain with an alpha/beta fold, not yet found in any other protein. The mode of RNA binding by this domain is hitherto unknown as is the rationale, if any, behind classification of anticodon-binding domains for different aaRSs. RESULTS: The crystal structure of Thermus thermophilus prolyl-tRNA synthetase (ProRSTT) in complex with tRNA(Pro) has been determined at 3.5 A resolution by molecular replacement using the native enzyme structure. One tRNA molecule, of which only the lower two-thirds is well ordered, is found bound to the synthetase dimer. The C-terminal anticodon-binding domain binds to the anticodon stem-loop from the major groove side. Binding to tRNA by ProRSTT is reminiscent of the interaction of class IIb enzymes with cognate tRNAs, but only three of the anticodon-loop bases become splayed out (bases 35-37) rather than five (bases 33-37) in the case of class IIb enzymes. The two anticodon bases conserved in all tRNA(Pro), G35 and G36, are specifically recognised by ProRSTT. CONCLUSIONS: For the synthetases possessing the class IIa anticodon-binding domain (ProRS, ThrRS and GlyRS, with the exception of HisRS), the two anticodon bases 35 and 36 are sufficient to uniquely identify the cognate tRNA (GG for proline, GU for threonine, CC for glycine), because these amino acids occupy full codon groups. The structure of ProRSTT in complex with its cognate tRNA shows that these two bases specifically interact with the enzyme, whereas base 34, which can be any base, is stacked under base 33 and makes no interactions with the synthetase. This is in agreement with biochemical experiments which identify bases 35 and 36 as major tRNA identity elements. In contrast, class IIb synthetases (AspRS, AsnRS and LysRS) have a distinct anticodon-binding domain that specifically recognises all three anticodon bases. This again correlates with the requirements of the genetic code for cognate tRNA identification, as the class IIb amino acids occupy half codon groups.

Amino Acid Sequence↗
PubMed1999

Elucidating the medium-resolution structure of ribosomal particles: an interplay between electron cryo-microscopy and X-ray crystallograhy.

BACKGROUND: Ribosomes are the universal cellular organelles that accomplish the translation of the genetic code into proteins. Electron cryo-microscopy (cryo-EM) has yielded fairly detailed three-dimensional reconstructions of ribosomes. These were used to assist in the determination of higher resolution structures by X-ray crystallography. RESULTS: Molecular replacement studies using cryo-EM reconstructions provided feasible packing schemes for crystals of ribosomes and their two subunits from Thermus thermophilus, and of the large subunits from Haloarcula marismortui. For the large subunits, these studies also confirmed the major heavy-atom sites obtained by single isomorphous replacement combined with anomalous diffraction (SIRAS) and by multiple isomorphous replacement combined with anomalous diffraction (MIRAS) at approximately 10 A. Although adequate starting phases could not be obtained for the small subunits, the crystals of which diffract to 3.0 A, cryo-EM reconstructions were indispensable for analyzing their 7.2 A multiple isomorphous replacement (MIR) map. This work indicated that the conformation of the crystallized small subunits resembles that seen within the 70S ribosomes. Subsequently, crystals of particles trapped in their functionally active state were grown. CONCLUSIONS: Single-particle cryo-EM can contribute to the progress of crystallography of non-symmetrical, large and flexible macromolecular assemblies. Besides confirming heavy-atom sites, obtained from flat or overcrowded difference Patterson maps, the cryo-EM reconstructions assisted in elucidating packing arrangements. They also provided tools for the identification of the conformation within the crystals and for the estimation of the level of inherent non-isomorphism.

Crystallography, X-Ray↗
PubMed2003

A versatile tRNA aminoacylation catalyst based on RNA.

Aminoacyl-tRNA synthetase (ARS) ribozymes have potential to develop a novel genetic coding system. Although we have previously isolated such a ribozyme that recognizes aromatic amino acids, it could not be used as a versatile catalyst due to its limited ability of aminoacylation to a particular tRNA used for the selection. To overcome this limitation, we used a combination of evolutionary and engineering approaches to generate an optimized ribozyme. The ribozyme, consisting of 45 nucleotides, displays a broad spectrum of activity toward various tRNAs. Most significantly, this ribozyme is able to exhibit multiple turnover activity and charge parasubstituted Phe analogs onto an engineered suppressor tRNA (tRNA(Asn)(CCCG)). Thus, it provides a useful and flexible tool for the custom synthesis of mischarged tRNAs with natural and nonnatural amino acids.

Amino Acids↗
PubMed2003

Epigenetic reprogramming in early mammalian development and following somatic nuclear transfer.

Epigenetic modifications of the genome play a significant role in the elaboration of the genetic code as established at fertilisation. These modifications affect early growth and development through their influence on gene expression especially on imprinted genes. Genome-wide epigenetic reprogramming in germ cells is essential in order to reset the parent-of-origin specific marking of imprinted genes, but may have a more general role in the restoration of totipotency in the early embryo. In a similar way, on somatic nuclear cloning, a differentiated cell must become 'reprogrammed' restoring totipotency in order to undergo development. Here we discuss the dynamic epigenetic reprogramming that takes place during normal development and highlight those areas with relevance to somatic nuclear cloning and the possibility of improving the efficiency of this process. We propose the concept of 'epigenetic checkpoints' for normal progression of development and the loss of totipotency.

Animals↗
PubMed2004

Alanyl-tRNA synthetase crystal structure and design for acceptor-stem recognition.

Early work on aminoacylation of alanine-specific tRNA (tRNA(Ala)) by alanyl-tRNA synthetase (AlaRS) gave rise to the concept of an early "second genetic code" imbedded in the acceptor stems of tRNAs. A single conserved and position-specific G:U base pair in the tRNA acceptor stem is the key identity determinant. Further understanding has been limited due to lack of a crystal structure of the enzyme. We determined a 2.14 A crystal structure of the 453 amino acid catalytic fragment of Aquifex aeolicus AlaRS. It contains the catalytic domain characteristic of class II synthetases, a helical domain with a hairpin motif critical for acceptor-stem recognition, and a C-terminal domain of a mixed alpha/beta fold. Docking of tRNA(Ala) on AlaRS shows critical contacts with the three domains, consistent with previous mutagenesis and functional data. It also suggests conformational flexibility within the C domain, which might allow for the positional variation of the key G:U base pair seen in some tRNA(Ala)s.

Alanine-tRNA Ligase↗
PubMed1999

Complement-resistance mechanisms of bacteria.

Despite more than a century of parallel research on bacteria and the complement system, relatively little is known of the mechanisms whereby pathogenic bacteria can escape complement-related opsonophagocytosis and direct killing. It is likely that pathogenicity in bacteria has arisen more accidentally than in viruses, and on the basis of selection from natural mutants rather than by outright stealing or copying of genetic codes from the host. In this review we will discuss complement resistance as one of the features that makes a bacterium a pathogen.

Antigenic Variation↗
PubMed2002

Incorporation of non-natural amino acids into proteins.

Chemical and biological diversity of protein structures and functions can be widely expanded by position-specific incorporation of non-natural amino acids carrying a variety of specialty side groups. After the pioneering works of Schultz's group and Chamberlin's group in 1989, noticeable progress has been made in expanding types of amino acids, in finding novel methods of tRNA aminoacylation and in extending genetic codes for directing the positions. Aminoacylation of tRNA with non-natural amino acids has been achieved by directed evolution of aminoacyl-tRNA synthetases or some ribozymes. Codons have been extended to include four-base codons or non-natural base pairs. Multiple incorporation of different non-natural amino acids has been achieved by the use of a different four-base codon for each tRNA. The combination of these novel techniques has opened the possibility of synthesising non-natural mutant proteins in living cells.

Acylation↗
PubMed2002

Sexual transmission of HIV-1 isolate showing G-->A hypermutation.

Retroviral genomes with a high frequency of G-->A mutations are thought to originate during reverse transcription (RT). Here we present a case report of an AIDS patient infected with a subtype F variant where extensive G-->A hypermutation (G-->A Hypm) sequences were found in the protease gene. This patient was failing HAART at the time the hypermutation was found. These sequences were basically encountered in the proviral compartment on two occasions and were persistently absent in the plasma viral population. The patient's viral genotype showed several mutations related to antiretroviral drug resistance in RT (T69N, M184V, T215F, K219Q) and protease (M36I, G48V, I54V, T63L, V82A) genes. The drug regimen was changed and the viral load dropped 0.9 Logs and CD4 count increased by 200 cells/ml. The hypermutation was not found any more in a 1-year follow up. The patient's wife was infected with a similar virus strain and G-->A Hypm sequences were also detected in the RT gene. This is the first report of sexual transmitted G-->A Hypermutation in HIV-1 and suggest that this phenomenon can be genetically coded by the viral RT molecule.

Acquired Immunodeficiency Syndrome↗

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