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PubMed1989

Glycine tRNA mutants with normal anticodon loop size cause -1 frameshifting.

Mutations in the acceptor stem, the 5-methyluridine-pseudouridine-cytidine (TFC) arm, and the anticodon of Salmonella tRNA2Gly can cause -1 frameshifting. The potential for standard base pairing between acceptor stem positions 1 and 72 is disrupted in the mutant sufS627. This disruption may interfere with the interaction of the tRNA with elongation factor-Tu.GTP or an as-yet-unspecified domain of the ribosome. The potential for standard base pairing in part of the TFC stem is disrupted in mutant sufS625. The nearly universal C-61 base of the TFC stem is altered in mutant sufS617, and the TFC loop is extended in mutant sufS605. These changes are expected to interfere with the stability of the TFC loop and its interaction with the D arm. The mutation in mutant sufS605, and possibly other mutants, alters nucleoside modification in the D arm. Three mutants, sufS601, sufS607, and sufS609, have a cytidine substituted for the modified uridine at position 34, the first anticodon position. None of the alterations grossly disrupts in-frame triplet decoding by the mutant tRNAs. The results show that -1 frameshifting in vivo can be caused by tRNAs with normal anticodon loop size and suggest that alternative conformational states of the mutant tRNAs may allow them to read a codon in frame or to shift reading frame.

Anticodon↗
PubMed2000

Pseudouridine in RNA: what, where, how, and why.

Pseudouridine (5-ribosyluracil) is a ubiquitous yet enigmatic constituent of structural RNAs (transfer, ribosomal, small nuclear, and small nucleolar). Although pseudouridine (psi) was the first modified nucleoside to be discovered in RNA, and is the most abundant, its biosynthesis and biological roles have remained poorly understood since its identification as a "fifth nucleoside" in RNA. Recently, a combination of biochemical, biophysical, and genetic approaches has helped to illuminate the structural consequences of psi in polyribonucleotides, the biochemical mechanism of U-->psi isomerization in RNA, and the role of modification enzymes (psi synthases) and box H/ACA snoRNAs, a class of eukaryotic small nucleolar RNAs, in the site-specific biosynthesis of psi. Through its unique ability to coordinate a structural water molecule via its free N1-H, psi exerts a subtle but significant "rigidifying" influence on the nearby sugar-phosphate backbone and also enhances base stacking. These effects may underlie the biological role of most (but perhaps not all) of the psi residues in RNA. Certain genetic mutants lacking specific psi residues in tRNA or rRNA exhibit difficulties in translation, display slow growth rates, and fail to compete effectively with wild-type strains in mixed culture. In particular, normal growth is severely compromised in an Escherichia coli mutant deficient in a pseudouridine synthase responsible for the formation of three closely spaced psi residues in the mRNA decoding region of the 23S rRNA. Such studies demonstrate that pseudouridylation of RNA confers an important selective advantage in a natural biological context.

Animals↗
PubMed1975

Studies on the mechanism of covalent incorporation of a lysergyl derivative to immunoglobulin peptides in vitro.

In vitro incubation of hyperimmune rabbit lymphoid cells with the hallucinogenic indole alkaloid, d-lysergic acid diethylamide (LSD), results in biosynthesis of modified, secretable immunoglobulin peptides. Modification involves covalent attachment of the lysergyl moiety to COOH-terminal portions of the peptides. Aalogous effects occur when cells are incubated in vitro in the presence of the non-hallucinogenic LSD analogue, d-lysergic acid, and N-[3H]-carboxymethyl-d-lysergamide. The phenomenon is reversed by tryptophan and is inhibited by puromycin and cycloheximide. In vitro attachment of the lysergyl moiety occurs in the presence of actinomycin D at levels which inhibit RNA synthesis. While LSD is not attached to intracellular tRNA, the drug binds to 80 S ribosomes from hyperimmune lymphoid cells with high affinity (K A equals 3.5 times 10-8 M-1). Similar binding occurs to nonimmune splenic ribosomes. Implications of these findings are discussed with respect to the degree of involvement of cellular protein translational mechanisms in the covalent attachment of the lysergyl moiety to low molecular weight immunoglobulin peptides.

Animals↗
PubMed2004

RNA-guided nucleotide modification of ribosomal and non-ribosomal RNAs in Archaea.

Archaea use ribonucleoprotein (RNP) machines similar to those found in the eukaryotic nucleolus to methylate ribose residues in nascent ribosomal RNA. The archaeal complex required for this 2'-O-ribose-methylation consists of the C/D box sRNA guide and three proteins, the core RNA-binding aL7a protein, the aNop56 protein and the methyltransferase aFib protein. These RNP machines were reconstituted in vitro from purified recombinant components, and shown to have methylation activity when provided with a simple target oligonucleotide, complementary to the sRNA guide sequence. To obtain a better understanding of the versatility and specificity of this reaction, the activity of reconstituted particles on more complex target substrates, including 5S RNA, tRNA(Gln) and 'double target' oligonucleotides that exhibit either direct or reverse complementarity to both the D' and D box guides, has been examined. The natural 5S and tRNA(Gln) substrates were efficiently methylated in vitro, as long as the complementarity between guide and target was about 10 base pairs in length, and lacked mismatches. Maximal activity of double guide sRNAs required that both methylation sites be present in cis on the target RNA.

Archaeal Proteins↗
PubMed1982

The effect of bisulfite-induced C to U transitions on aminoacylation of Escherichia coli glycine tRNA.

The effect of bisulfite-induced C to U transitions on the aminoacylation of highly purified Escherichia coli tRNAgly3,gcc has been studied. On treatment with 3.2 M NaHSO3 at 25 degrees C and pH 5.8, C to U transitions occurred at 4 of the 21 cytosine residues during the first 16 h. After about 12 h, 2 additional unidentified residues begin to react. From these data and theoretical consideration, we conclude: 1) E. coli tRNAgly3,gcc exists in the native conformation under the conditions of the reaction; 2) its ordered structure is similar to yeast tRNAPhe; 3) its anticodon loop is flexible in solution. During the reaction, loss of glycine acceptor activity followed first order kinetics with a t1/2 = 1.8 h. After modification for 1.25 h and aminoacylation (61% glycine acceptor activity remaining), the aminoacylated fraction was isolated. The fractional change at each of the reactive residues in the unfractionated mixture (f) and the aminoacylated fraction (p) was measured. The results were: C35, f = 0.33, p = 0.19; C36, f = 0.25, p = 0.10; C74, f = 0.28, p = 0.26; C75, f = 0.25, p = 0.20. From these data, the Modulation Constant for each reactive residue was calculated from the equation M = (f - p)/f(1 - p): M35 (anticodon) = 0.52; M36 (anticodon) = 0.67; M74 (CCA end) = 0.10; M75 (CCA end) = 0.25. These values, which are based on the assumptions that the C to U reactions occur independently and that the effect of each change on the acceptor activity is an independent event, express the fractional loss in activity that would occur from a C to U change at the residue in question by itself. From these results, we conclude: 1) approximately 80% of the observed inactivation was due to changes in the anticodon; 2) neither of these anticodon residues (C35 and C36) is essential for aminoacylation; 3) a C to U change at C75 (CCA end) has a small effect on aminoacylation; 4) a C to U change at C74 (CCA end) has little or no effect on aminoacylation.

Cytosine↗
PubMed1999

RNA editing.

The term RNA editing describes those molecular processes in which the information content is altered in an RNA molecule. To date such changes have been observed in tRNA. rRNA and mRNA molecules of eukaryotes, but not prokaryotes. The demonstration of RNA editing in prokaryotes may only be a matter of time, considering the range of species in which the various RNA editing processes have been found. RNA editing occurs in the nucleus, as well as in mitochondria and plastids, which are thought to have evolved from prokaryotic-like endosymbionts. Most of the RNA editing processes, however, appear to be evolutionarily recent acquisitions that arose independently. The diversity of RNA editing mechanisms includes nucleoside modifications such as C to U and A to I deaminations, as well as non-templated nucleotide additions and insertions. RNA editing in mRNAs effectively alters the amino acid sequence of the encoded protein so that it differs from that predicted by the genomic DNA sequence.

Amino Acid Sequence↗
PubMed2004

RETRACTED: A new strategy for the synthesis of glycoproteins.

Posttranslational modifications of proteins regulate many biological processes, including metabolism, signal transduction, and gene expression. The synthetic challenges associated with generating homogeneous populations of selectively modified proteins, however, have hindered detailed studies of the effects of these modifications on protein structure and function. Here, we report an approach to the cotranslational synthesis of selectively glycosylated proteins in which the modified amino acid is genetically encoded. We show that myoglobin containing beta-N-acetylglucosamine (GlcNAc)-serine at a defined position can be expressed in Escherichia coli in good yield and with high fidelity. The beta-GlcNAc moiety can be recognized by a saccharide-binding protein, or subsequently modified with a galactosyltransferase to build more complex carbohydrates. This approach should be generally applicable to other posttranslational modifications such as protein phosphorylation, acetylation, and methylation.

Acetylation↗
PubMed1999

Substrate binding and catalysis by ribonuclease P from cyanobacteria and Escherichia coli are affected differently by the 3' terminal CCA in tRNA precursors.

We have studied the effect of the 3' terminal CCA sequence in precursors of tRNAs on catalysis by the RNase P RNA or the holoenzyme from the cyanobacterium Synechocystis sp. PCC 6803 in a completely homologous system. We have found that the absence of the 3' terminal CCA is not detrimental to activity, which is in sharp contrast to what is known in other bacterial systems. We have found that this is also true in other cyanobacteria. This situation correlates with the anomalous structure of the J15/16 loop in cyanobacteria, which is an important loop in the CCA interaction in Escherichia coli RNase P, and with the fact that cyanobacteria do not code the CCA sequence in the genome but add it posttranscriptionally. Modification of nucleotides 330-332 in the J15/16 loop of Synechocystis RNase P RNA from GGU to CCA has a modest effect on kcat for CCA-containing substrates and has no effect on cleavage-site selection. We have developed a direct physical assay of the interaction between RNase P RNA and its substrate, which was immobilized on a filter, and we have determined that Synechocystis RNase P RNA binds with better affinity the substrate lacking CCA than the substrate containing it. Our results indicate a mode of substrate binding in RNase P from cyanobacteria that is different from binding in other eubacteria and in which the 3' terminal CCA is not involved.

Base Sequence↗
PubMed2005

Chemical engineering of the peptidyl transferase center reveals an important role of the 2'-hydroxyl group of A2451.

The main enzymatic reaction of the large ribosomal subunit is peptide bond formation. Ribosome crystallography showed that A2451 of 23S rRNA makes the closest approach to the attacking amino group of aminoacyl-tRNA. Mutations of A2451 had relatively small effects on transpeptidation and failed to unequivocally identify the crucial functional group(s). Here, we employed an in vitro reconstitution system for chemical engineering the peptidyl transferase center by introducing non-natural nucleosides at position A2451. This allowed us to investigate the peptidyl transfer reaction performed by a ribosome that contained a modified nucleoside at the active site. The main finding is that ribosomes carrying a 2'-deoxyribose at A2451 showed a compromised peptidyl transferase activity. In variance, adenine base modifications and even the removal of the entire nucleobase at A2451 had only little impact on peptide bond formation, as long as the 2'-hydroxyl was present. This implicates a functional or structural role of the 2'-hydroxyl group at A2451 for transpeptidation.

Adenine↗
PubMed1994

1H NMR structure and biological studies of the His23-->Cys mutant nucleocapsid protein of HIV-1 indicate that the conformation of the first zinc finger is critical for virus infectivity.

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1), which has key functions in the virus life cycle, possesses two zinc fingers of the CX2CX4HX4C type characterized by three successive loops containing a tetrahedrally coordinated zinc atom. The replacement of any cysteine by a serine in either finger has been shown to result in the production of noninfectious viruses, probably by impairing the biological functions of NCp7. In order to more precisely elucidate the structural role of the zinc finger motif, His23 was replaced by Cys in the proximal finger of the peptide (13-64)NCp7 which retains NCp7 activities in vitro. The peptide Cys23(13-64)NCp7 was synthesized by solid phase and studied by 2D 1H NMR and molecular modeling. The His to Cys modification causes important structural modifications of the N-terminal zinc finger which impair the spatial proximity of the two zinc fingers as shown by the disappearance of several interresidue NOEs. The side chains of Val13, Lys14, Phe16, Thr24, Ala25, Trp37, Gln45, and Met46, which are thought to be involved in nucleic acid recognition, are no longer found clustered in the Cys23(13-64)NCp7 mutant as they are in the wild-type NCp7 structure. In vitro, Cys23(13-64)NCp7 is unable to tightly interact with the viral RNA or replication primer tRNA(Lys,3). The Cys23(NCp7) mutation was introduced into an infectious HIV-1 molecular clone, and virions produced upon DNA transfection into cells were analyzed for their viral protein and RNA compositions as well as for their infectivity. Results show that, while the Cys23(NCp7) mutation does not impair virion production, viruses contain a low amount of degraded viral RNA and are not infectious. These findings suggest that a bona fide conformation of the HIV-1 NCp7 is critical for the packaging of viral RNA, its stability in virions, and virus infectivity.

Amino Acid Sequence↗
PubMed2003

Programmed translational -1 frameshifting on hexanucleotide motifs and the wobble properties of tRNAs.

Programmed -1 ribosomal frameshifting, involving tRNA re-pairing from an AAG codon to an AAA codon, has been reported to occur at the sequences CGA AAG and CAA AAG. In this study, using the recoding region of insertion sequence IS3, we have investigated the influence on frameshifting in Escherichia coli of the first codon of this type of motif by changing it to all other NNA codons. Two classes of NNA codons were distinguished, depending on whether they favor or limit frameshifting. Their degree of shiftiness is correlated with wobble propensity, and base 34 modification, of their decoding tRNAs. A more flexible anticodon loop very likely makes the tRNAs with extended wobble more prone to liberate the third codon base, A, for re-pairing of tRNALys in the -1 frame.

Base Sequence↗
PubMed1975

Isoaccepting lysine transfer ribonucleic acid species of Pseudomonas aeruginosa.

Pseudomonas aeruginosa tRNA was treated with iodine, CNBr and N-ethylmaleimide, three thionucleotide-specific reagents. Reaction with iodine resulted in extensive loss of acceptor activity by lysine tRNA, glutamic acid tRNA, glutamine tRNA, serine tRNA and tyrosine tRNA. CNBr treatment resulted in high loss of acceptor ability by lysine tRNA, glutamic acid tRNA and glutamine tRNA. Only the acceptor ability of tyrosine tRNA was inhibited up to 66% by N-ethylmaleimide treatment, a reagent specific for 4-thiouridine. By the combined use of benzoylated DEAE-cellulose and DEAE-Sephadex columns, lysine tRNA of Ps. aeruginosa was resolved into two isoaccepting species, a major, tRNA Lys1 and a minor, tRNALys1. Co-chromatography of 14C-labelled tRNALys1 and 3H-labelled tRNALys2 on benzoylated DEAE-cellulose at pH 4.5 gave two distinct, non-superimposable profiles for the two activity peaks, suggesting that they were separate species. The acceptor activity of these two species was inhibited by about 95% by iodine and CNBr. Both the species showed equal response to codons AAA and AAG and also for poly(A) and poly(A1,G1) suggesting that the anticodon of these species was UUU. Chemical modification of these two species by iodine did not inhibit the coding response. The two species of lysine of Ps. aeruginosa are truly redundant in that they are indistinguishable either by chemical modification or by their coding response.

Anticodon↗
PubMed1998

Recent developments in streptogramin research.

The streptogramins are a class of antibiotics remarkable for their antibacterial activity and their unique mechanism of action. These antibiotics are produced naturally, but the therapeutic use of the natural compounds is limited because they do not dissolve in water. New semisynthetic derivatives, in particular the injectable streptogramin quinupristin/dalfopristin, offer promise for treating the rising number of infections that are caused by multiply resistant bacteria. The streptogramins consist of two structurally unrelated compounds, group A and group B. The group A compounds are polyunsaturated macrolactones: the group B compounds are cyclic hexadepsipeptides. Modifications of the group B components have been mainly performed on the 3-hydroxypicolinoyl, the 4-dimethylaminophenylalanine and the 4-oxo pipecolinic residues. Semi-synthesis on this third residue led to the water-soluble derivative quinupristin. Water-soluble group A derivatives were obtained by Michael addition of aminothiols to the dehydroproline ring of pristinamycin IIA. Followed by oxidation of the intermediate sulfide into the sulfone derivatives (i.e., dalfopristin). Water-soluble derivatives (both group A and group B) can now be obtained at the industrial scale. Modified group B compounds are now also being produced by mutasynthesis, via disruption of the papA gene. Mutasynthesis has proved particularly useful for producing PIB, the group B component of the oral streptogramin RPR 106972. The streptogramins inhibit bacterial growth by disrupting the translation of mRNA into protein. Both the group A and group B compounds bind to the peptidyltransferase domain of the bacterial ribosome. The group A compounds interfere with the elongation of the polypeptide chain by preventing the binding of aa-tRNA to the ribosome and the formation of peptide bonds, while the B compounds stimulate the dissociation of the peptidyl-tRNA and may also interfere with the release of the completed polypeptide by blocking its access to the channel through which it normally leaves the ribosome. The synergy between the group A and group B compounds appears to result from an enhanced affinity of the group B compounds for the ribosome. Apparently, the group A compound induces a conformational change such that B compound binds with greater affinity. The natural streptogramins are produced as mixtures of the group A and B compounds, the combination of which is a more potent antibacterial agent than either type of compound alone. Whereas the type A or type B compound alone has, in vitro and in animal models of infection, a moderate bacteriostatic activity, the combination of the two has strong bacteriostatic activity and often bactericidal activity. Minimal inhibitory concentrations of quinupristin/dalfopristin range from 0.20 to 1 mg/l for Streptococcus pneumonae, from 0.25 to 2 mg/l for Staphylococcus aureus and from 0.50 to 4 for Enterococcus faecium, the principal target organisms of this drug. Quinupristin/dalfopristin also has activity against mycoplasmas, Neisseria gonorrhoeae, Haemophilus influenz, Legionella spp. and Moraxella catarrhalis. Bacteria develop resistance to the streptogramms by ribosomal modification, by producing inactivating enzymes, or by causing an efflux of the antibiotic. Dimethylation of an adenine residue in rRNA, a reaction that is catalyzed by a methylase encoded by the erm gene class, affects the binding of group B compounds (as well as the macrolides and lincosamides; hence, MLSB resistance), but group A and B compounds usually maintain their synergy and their bactericidal effect against MLSB-resistant strains. erm genes are widespread both geographically and throughout numerous bacterial genera. Several types of enzymes (acetyltransferases, hydrolases) have been identified that inactivate the group A or the group B compounds. Genes involved in streptogramin efflux have so far been found only in staphylococci, particularly in coagulase-negative species

Animals↗
PubMed1980

The structure and aminoacylation of a temperature-sensitive tRNATrp (Escherichia coli).

A temperature-sensitive (t.s.) tRNATrp from Escherichia coli has a single base change from the wild type (w.t.) species, which results in the loss of a base pair at the bottom of the CCA stem of the cloverleaf structure. Thermodynamic studies on this t.s. tRNA show that it is more susceptible to denaturation than the w.t. due to a larger change in the entropy of denaturation. Correlated with this thermodynamic result is the finding that the denatured t.s. tRNA's T psi C loop is more susceptible to digestion by T1 RNase, suggesting that it has greater freedom than the corresponding structure on the denatured w.t. molecule. In contrast, the native form of the t.s. tRNATrp is very similar to the w.t. with regard to aminoacylation, T1 RNase susceptibility, and column chromatographic mobility, despite the fact that it necessarily has one less base pair. In addition, the well known denaturation-dependent shift in column chromatographic mobility, which is observed for both the t.s. and w.t. molecules, depends on a modification in the anticodon loop, since tRNATrp lacking that modification does not shift when denatured. Thus, though it is not usually thought to be implicated, denaturation probably affects the conformation of the anticodon loop. The lethal phenotype of the mutant at high temperature, defective attenuation of the tryptophan biosynthetic operon in the mutant, and some aspects of the denatured state are clarified by these findings.

Acylation↗
PubMed1998

Modified nucleotides of tRNAPro restrict interactions in the binary primer/template complex of M-MuLV.

In all retroviruses, reverse transcription is primed by a cellular tRNA, which is base-paired through its 3'-terminal 18 nucleotides to a complementary sequence on the viral RNA genome termed the primer binding site (PBS). Evidence for specific primer-template interactions in addition to this standard interaction has recently been demonstrated for several retroviruses. Here, we used chemical and enzymatic probing to investigate the interactions between Moloney murine leukemia virus (M-MuLV) RNA and its natural primer tRNAPro. The existence of extended interactions was further tested by comparing the viral RNA/tRNAPro complex with simplified complexes in which viral RNA or tRNA were reduced to the 18 nt of the PBS or to the complementary tRNA sequence. These data, combined with computer modeling provide important clues on the secondary structure and three-dimensional folding of the M-MuLV RNA/tRNAPro complex. In contrast with other retroviruses, we found that the interaction between tRNAPro and the M-MuLV RNA template is restricted to the standard PBS interaction. In this binary complex, the viral RNA is highly constrained and the rest of tRNAPro is rearranged, with the exception of the anticodon arm, leading to a very compact structure. Unexpectedly, when a synthetic tRNAPro lacking the post-transcriptional modifications is substituted for the natural tRNAPro primer, the interactions between the primer and the viral RNA are extended. Hence, our data suggest that the post-transcriptional modifications of natural tRNAPro prevent additional contacts between tRNAPro and the U5 region of M-MuLV RNA.

Models, Molecular↗
PubMed2005

Ribosomal RNA in Alzheimer disease is oxidized by bound redox-active iron.

Oxidative modification of cytoplasmic RNA in vulnerable neurons is an important, well documented feature of the pathophysiology of Alzheimer disease. Here we report that RNA-bound iron plays a pivotal role for RNA oxidation in vulnerable neurons in Alzheimer disease brain. The cytoplasm of hippocampal neurons showed significantly higher redox activity and iron(II) staining than age-matched controls. Notably, both were susceptible to RNase, suggesting a physical association of iron(II) with RNA. Ultrastructural analysis further suggested an endoplasmic reticulum association. Both rRNA and mRNA showed twice the iron binding as tRNA. rRNA, extremely abundant in neurons, was considered to provide the greatest number of iron binding sites among cytoplasmic RNA species. Interestingly, the difference of iron binding capacity disappeared after denaturation of RNA, suggesting that the higher order structure may contribute to the greater iron binding of rRNA. Reflecting the difference of iron binding capacity, oxidation of rRNA by the Fenton reaction formed 13 times more 8-hydroxyguanosine than tRNA. Consistent with in situ findings, ribosomes purified from Alzheimer hippocampus contained significantly higher levels of RNase-sensitive iron(II) and redox activity than control. Furthermore, only Alzheimer rRNA contains 8-hydroxyguanosine in reverse transcriptase-PCR. Addressing the biological significance of ribosome oxidation by redox-active iron, in vitro translation with oxidized ribosomes from rabbit reticulocyte showed a significant reduction of protein synthesis. In conclusion these results suggest that rRNA provides a binding site for redox-active iron and serves as a redox center within the cytoplasm of vulnerable neurons in Alzheimer disease in advance of the appearance of morphological change indicating neurodegeneration.

Alzheimer Disease↗
PubMed2003

Lysyl-tRNA synthetase from Bacillus stearothermophilus: the Trp314 residue is shielded in a non-polar environment and is responsible for the fluorescence changes observed in the amino acid activation reaction.

Three Trp variants of lysyl-tRNA synthetase from Bacillus stearothermophilus, in which either one or both of the two Trp residues within the enzyme (Trp314 and Trp332) were substituted by a Phe residue, were produced by site-directed mutagenesis without appreciable loss of catalytic activity. The following two phenomena were observed with W332F and with the wild-type enzyme, but not with W314F: (1) the addition of L-lysine alone decreased the protein fluorescence of the enzyme, but the addition of ATP alone did not; (2) the subsequent addition of ATP after the addition of excess L-lysine restored the fluorescence to its original level. Fluorometry under various conditions and UV-absorption spectroscopy revealed that Trp314, which was about 20A away from the lysine binding site and was shielded in a non-polar environment, was solely responsible for the fluorescence changes of the enzyme in the L-lysine activation reaction. Furthermore, the microenvironmental conditions around the residue were made more polar upon the binding of L-lysine, though its contact with the solvent was still restricted. It was suggested that Trp314 was located in a less polar environment than was Trp332, after comparison of the wavelengths at the peaks of fluorescence emission and of the relative fluorescence quantum yields. Trp332 was thought, based on the fluorescence quenching by some perturbants and the chemical modification with N-bromosuccinimide, to be on the surface of the enzyme, whereas Trp314 was buried inside. The UV absorption difference spectra induced by the L-lysine binding indicated that the state of Trp314, including its electrostatic environment, changed during the process, but Trp332 did not change. The increased fluorescence from Trp314 at acidic pH compared with that at neutral pH suggests that carboxylate(s) are in close proximity to the Trp314 residue.

Amino Acid Substitution↗
PubMed1989

Identification of the N-tosyl-L-phenylalanyl chloromethylketone modification site in Thermus thermophilus elongation factor Tu.

EF-Tu from Thermus thermophilus was first labelled with N-[14C]tosyl-L-phenylalaninechloromethylketone and then cleaved by the combined action of CNBr and trypsin. The resulting peptides were separated by reversed-phase HPLC. Analysis of the isolated, labelled peptide led to the identification of a sequence which was identical to residues 76-88 in T. thermophilus EF-Tu. The TPCK reactive site is at Cys-82. Kinetic measurements of the incorporation of TPCK into native EF-Tu and EF-Tu nicked at position Arg-59 were performed. The results provide evidence that the cleavage of the peptide bond between Arg-59 and Gly-60 does not lead to a dramatic conformational change of EF-Tu at the aa-tRNA binding site.

Amino Acid Chloromethyl Ketones↗