Influence of the last amino acid in the nascent peptide on EF-Tu during decoding

The last two amino acids of the nascent peptide at the ribosomal P-site influence the efficiency of termination readthrough at the stop codon UGA (Mottagui-Tabar et al (1994) EMBO J 13, 249–257; Björnsson et al (1996) EMBO J 15, 1696–1704). Here we analyze this effect on readthrough by wild type or a UGA suppressor form (Su9) of tRNA^Trp by varying the codons at positions −1 and −2 at the 5′ side of UGA. Strains with wild-type or mutant (ArBr) forms of elongation factor Tu (EF-Tu) were analyzed (Vijgenboom et al (1985) EMBO J 4, 1049–1052). The effect on readthrough by changing these −1 and −2 codons is different on the two forms of tRNA^Trp and is also dependent on the structure of EF-Tu. Readthrough by the tRNA^Trp-derived suppressor, but not wild-type tRNA^Trp, is sensitive to the van der Waals volume of the last amino acid in the nascent peptide. Together with mutant EF-Tu, both forms of tRNA^Trp are sensitive. The data suggest that the C-terminal amino acid in the nascent peptide is in a functional interaction with the EF-Tu ternary complex. This interaction is changed by mutation in tRNA^Trp at position 24 or in EF-Tu at position 375. No indication of a changed interaction between the mutant EF-Tu and the penultimate amino acid could be found. Mutant forms of RF2 (Mikuni et al (1991) Biochimie 73, 1509–1516) and ribosomal proteins S4 and S12 (Fáxen et al (1988) J Bacteriol 170, 3756–3760) were found not to be altered in sensitivity to the last two amino acids in the nascent peptide.     

The C-terminal amino acid sequence of nascent peptide is a major determinant of SsrA tagging at all three stop codons

Recent studies on endogenous SsrA-tagged proteins have revealed that the tagging could occur at a position corresponding to the normal termination codon. During the study of SsrA-mediated Lacl tagging (Abo et al., EMBO J, 2000 19:3762-3769), we found that a variant Lacl (Lacl deltaC1) lacking the last C-terminal amino acid residue is efficiently tagged in a stop codon-dependent manner. SsrA tagging of Lacl deltaC1 occurred efficiently without Lacl binding to the lac operators at any one of three stop codons. The C-terminal (R)LESG peptide of Lacl deltaC1 was shown to trigger the SsrA tagging of an unrelated protein (CRP) when fused to its C terminus. Mass spectrometry analysis of the purified fusion proteins revealed that SsrA tagging occurs at a position corresponding to the termination codon. The alteration of the amino acid sequence but not the nucleotide sequence of the C-terminal portion eliminated the tagging. We also showed that the tagging-provoking sequences cause an efficient translational readthrough at UGA but not UAA codons. In addition, we found that C-terminal dipeptides known to induce an efficient translation readthrough could cause an efficient tagging at stop codons. We conclude that the amino acid sequence of nascent polypeptide prior to stop codons is a major determinant for the SsrA tagging at all three stop codons.     

The amino acid sequence of a hypothalamic peptide, neurotensin.

The amino acid sequence of neurotensin, a hypotensive peptide isolated from acid-acetone extracts of bovine hypothalami, has been established as less than Glu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-Oh. (The nomenclature and symbols follow the suggestions of the IUPAC-IUB Commission on Biochemical Nomenclature (1972) J. Biol. Chem. 247, 977). This was accomplished by sequence analyses performed on the intact peptide as well as its isolated tryptic, chymotryptic, and papain-generated fragments. The results of enzymic hydrolyses were consistent with the specificities of the enzymes used and indicated that all of the amino acids are unsubstituted and in the L configuration. The absence of non-amino acid constituents was further supported by analyses of electrophoretic mobility-molecular weight relationships of neurotensin and its fragments.     

Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli

The immutability of the genetic code has been challenged with the successful reassignment of the UAG stop codon to non-natural amino acids in Escherichia coli. In the present study, we demonstrated the in vivo reassignment of the AGG sense codon from arginine to l-homoarginine. As the first step, we engineered a novel variant of the archaeal pyrrolysyl-tRNA synthetase (PylRS) able to recognize l-homoarginine and l-N⁶-(1-iminoethyl)lysine (l-NIL). When this PylRS variant or HarRS was expressed in E. coli, together with the AGG-reading tRNA^Pyl_CCU molecule, these arginine analogs were efficiently incorporated into proteins in response to AGG. Next, some or all of the AGG codons in the essential genes were eliminated by their synonymous replacements with other arginine codons, whereas the majority of the AGG codons remained in the genome. The bacterial host''s ability to translate AGG into arginine was then restricted in a temperature-dependent manner. The temperature sensitivity caused by this restriction was rescued by the translation of AGG to l-homoarginine or l-NIL. The assignment of AGG to l-homoarginine in the cells was confirmed by mass spectrometric analyses. The results showed the feasibility of breaking the degeneracy of sense codons to enhance the amino-acid diversity in the genetic code.     

Insights on the amino acid side-chain interactions of a synthetic T-cell determinant.

The effect of single amino acid substitutions at positions 18 and 20 on the T-cell determinant (TD) character of peptide p12-26 from lambda repressor protein and on its recognition by a monoclonal antibody was studied by means of 40 synthetic peptides of a length of 15 amino acids. ELISA competition experiments showed that the identity of amino acid at position 20 is very important for antibody recognition, whereas that of amino acid at position 18 is much less important. In contrast, both Leu 18 and Ala 20 are important residues in defining the TD character of peptide p12-26. The most tolerated replacements, ordered in increasing disrupting power are: Ala 20 by Cys, Ser or Gly and Leu 18 by Ile or Val. Any other amino acid replacement completely abolishes the TD capacity of peptide p12-26. The peptides used in this study were synthesized using a multiple solid-phase peptide synthesizer newly designed. Their purity was very high as shown by amino acid sequence experiments.     

Selection for efficient translation initiation biases codon usage at second amino acid position in secretory proteins

The definition of a typical sec-dependent bacterial signal peptide contains a positive charge at the N-terminus, thought to be required for membrane association. In this study the amino acid distribution of all Escherichia coli secretory proteins were analysed. This revealed that there was a statistically significant bias for lysine at the second codon position (P2), consistent with a role for the positive charge in secretion. Removal of the positively charged residue P2 in two different model systems revealed that a positive charge is not required for protein export. A well-characterized feature of large amino acids like lysine at P2 is inhibition of N-terminal methionine removal by methionyl amino-peptidase (MAP). Substitution of lysine at P2 for other large or small amino acids did not affect protein export. Analysis of codon usage revealed that there was a bias for the AAA lysine codon at P2, suggesting that a non-coding function for the AAA codon may be responsible for the strong bias for lysine at P2 of secretory signal sequences. We conclude that the selection for high translation initiation efficiency maybe the selective pressure that has led to codon and consequent amino acid usage at P2 of secretory proteins.     

A single amino acid in the second transmembrane domain of GABA rho subunits is a determinant of the response kinetics of GABAC receptors.

The rho subunits that constitute the gamma-aminobutyric acid (GABA)C receptors of retinal neurons form a unique subclass of ligand-gated chloride channels that give rise to sustained GABA-evoked currents that exhibit slow offset (deactivation) kinetics. We exploited this property to examine the molecular mechanisms that govern the disparate response kinetics and pharmacology of perch GABA rho1B and rho2A subunits expressed in Xenopus oocytes. Using a combination of domain swapping and site-directed mutagenesis, we identified the residues at amino acid position 320 in the second transmembrane domain as an important determinant of the receptor kinetics of GABAC receptors. When the site contains a proline residue, as in wild-type rho1 subunits, the receptor deactivates slowly; when serine occupies the site, as in wild-type rho2 subunits, the time course of deactivation is more rapid. In addition, we found that the same site also altered the pharmacology of GABA rho receptors, e.g., when the serine residue of the rho2A receptor was changed to proline, the response of the mutant receptor to imidazole-4-acetic acid (I4AA) mimicked that of the rho1B receptor. However, despite gross changes in receptor pharmacology, the apparent binding affinity for the drug was not significantly altered. These findings provide further evidence that the second transmembrane domain is involved in the gating mechanism that governs the response properties of the various rho receptor subunits. It is noteworthy that the proline residue in native rho1 subunits and the serine residue of rho2 subunits are well conserved in all species, a good indication that the presence of multiple GABA rho subunits serves to generate GABAC receptors that display the wide range of response kinetics observed on various types of retinal neurons.     

The universal dinucleotide asymmetry rules in DNA and the amino acid codon choice

Summary Natural DNA sequences were recently found to contain distinct nearest neighbor patterns. Hetero-dinucleotides were demonstrated to appear consistently more (less) than their mirror-image counterparts. This paper shows that this assymmetric behavior does not stem from the coding requirements of the DNA. It also shows some codon patterns in prokaryotic and eukaryotic genomes which came up in the course of this work.     

The evolution of biased codon and amino acid usage in nematode genomes

Despite the degeneracy of the genetic code, whereby different codons encode the same amino acid, alternative codons and amino acids are utilized nonrandomly within and between genomes. Such biases in codon and amino acid usage have been demonstrated extensively in prokaryote genomes and likely reflect a balance between the action of mutation, selection, and genetic drift. Here, we quantify the effects of selection and mutation drift as causes of codon and amino acid-usage bias in a large collection of nematode partial genomes from 37 species spanning approximately 700 Myr of evolution, as inferred from expressed sequence tag (EST) measures of gene expression and from base composition variation. Average G + C content at silent sites among these taxa ranges from 10% to 63%, and EST counts range more than 100-fold, underlying marked differences between the identities of major codons and optimal codons for a given species as well as influencing patterns of amino acid abundance among taxa. Few species in our sample demonstrate a dominant role of selection in shaping intragenomic codon-usage biases, and these are principally free living rather than parasitic nematodes. This suggests that deviations in effective population size among species, with small effective sizes among parasites, are partly responsible for species differences in the extent to which selection shapes patterns of codon usage. Nevertheless, a consensus set of optimal codons emerges that is common to most taxa, indicating that, with some notable exceptions, selection for translational efficiency and accuracy favors similar sets of codons regardless of the major codon-usage trends defined by base compositional properties of individual nematode genomes.     

Amino-acyl tXNA as inhibitors or amino acid donors in peptide synthesis

Xenobiotic nucleic acids (XNAs) offer tremendous potential for synthetic biology, biotechnology, and molecular medicine but their ability to mimic nucleic acids still needs to be explored. Here, to study the ability of XNA oligonucleotides to mimic tRNA, we synthesized three L-Ala-tXNAs analogs. These molecules were used in a non-ribosomal peptide synthesis involving a bacterial Fem transferase. We compared the ability of this enzyme to use amino-acyl tXNAs containing 1′,5′-anhydrohexitol (HNA), 2′-fluoro ribose (2′F-RNA) and 2′-fluoro arabinose. L-Ala-tXNA containing HNA or 2′F-RNA were substrates of the Fem enzyme. The synthesis of peptidyl-XNA and the resolution of their structures in complex with the enzyme show the impact of the XNA on protein binding. For the first time we describe functional tXNA in an in vitro assay. These results invite to test tXNA also as substitute for tRNA in translation.     

Agarose-chymotrypsin as a catalyst for peptide and amino acid ester synthesis in organic media

Summary Chymotrypsin immobilised within agarose gels by multi-point covalent attachment is a useful catalyst for peptide or amino acid ester synthesis in mainly organic media. Moist gel beads (optionally containing one reactant) may be suspended in organic phases based on ethyl acetate, 1,1,1-trichloroethane or pentan-3-one.     

Engineering dehydrated amino acid residues in the antimicrobial peptide nisin.

The small antimicrobial peptide nisin, produced by Lactococcus lactis, contains the uncommon amino acid residues dehydroalanine and dehydrobutyrine and five thio ether bridges. Since these structures are posttranslationally formed from Ser, Thr, and Cys residues, it is feasible to study their role in nisin function and biosynthesis by protein engineering. Here we report the development of an expression system for mutated nisin Z (nisZ) genes, using nisin A producing L. lactis as a host. Replacement by site-directed mutagenesis of the Ser-5 codon in nisZ by a Thr codon, led to a mutant with a dehydrobutyrine instead of a dehydroalanine residue at position 5, as shown by NMR. Its antimicrobial activity was 2-10-fold lower relative to wild-type nisin Z, depending on the indicator strain used. In another mutagenesis study a double mutation was introduced in the nisZ gene by replacing the codons for Met-17 and Gly-18 by codons for Gln and Thr, respectively, as in the third lanthionine ring of the related antimicrobial peptide subtilin from Bacillus subtilis. This resulted in the simultaneous production of two mutant species, one containing a Thr residue and the other containing a dehydrobutyrine residue at position 18, both having different bacteriocidal properties.     

Alanine is the main second amino acid in vertebrate proteins and its coding entails increased use of the rare codon GCG

The occurrence of a main amino acid at the start of proteins from various vertebrate species is reported. Computer analyses of protein-coding sequences showed that alanine occurs in one out of five cases as the second amino acid in proteins from seven mammals (including man), one amphibian, one bird and two fishes. We also show that the alanine codon GCG occurs >3-fold more often as second codon than in general, i.e. GCG was overrepresented. Options to explain the abundance of alanine as second amino acid may already exist, but the overrepresentation of GCG was harder to explain because GCG was classically a rare codon. However, based on similarities with the published translation-initiation enhancer sequence CCGGCGG, which has complementarity with 18S ribosomal RNA, a theoretical role of GCG in translation initiation is suggested. Namely, GCG is proposed to be part of a sequence with potential for base-pairing with 18S ribosomal RNA.     

Sugar amino acid based peptide epoxyketones as potential proteasome inhibitors

This paper describes the synthesis and biological evaluation of nine epoxomicin-derived sugar amino acid containing peptide epoxyketones. The title compounds are assembled from six sugar amino acid dipeptide isosteres and are synthesized using solution-phase peptide synthesis protocols. Although neither of the compounds displays inhibitory activity towards any of the proteasome active sites, our approach holds promise towards the development of structurally new proteasome inhibitors. It is likely that the central sugar amino acid dipeptide isoster needs to be designed such that it closely resemble dipeptides at position P2 and P3 in proteasome substrates inhibitors, such as the Thr-Ile dipeptide present in the lead compound, epoxomicin.     

The effect of context on synonymous codon usage in genes with low codon usage bias.

The effect of neighbouring bases on the usage of synonymous codons in genes with low codon usage bias in yeast and E. coli is examined. The codon adaptation index is employed to identify a group of genes in each organism with low codon usage bias, which are likely to be weakly expressed. A similar pattern is found in complementary sequences with respect to synonymous usage of A vs G or of U vs C. It is suggested that this may reflect an effect of context on mutation rates in weakly expressed genes.     

Glutamine-stimulated amino acid and peptide incorporation in Bacteroides melaninogenicus.

The uptake of a number of amino acids and dipeptides by cells and spheroplasts of Bacteroides melaninogenicus was stimulated by the presence of glutamine; 50 mM glutamine induced maximum uptake of glycine or alanine, and glutamine stimulated the uptake of glycine over a wide concentration range (0.17 to 170 mM). Glutamine stimulated the uptake of the dipeptides glycylleucine and glycylproline at significantly faster rates compared with glycine and leucine. The amino acids whose uptake was stimulated by glutamine were incorporated into trichloroacetic acid-precipitable material, and the inclusion of chloramphenicol or puromycin did not affect this incorporation. The uptake of glutamine by cells was concentration dependent. In contrast, in the absence of chloramphenicol 79% of the glutamine taken up by cells supplied with a high external concentration (4.4 mM) was trichloroacetic acid soluble. Glutamate and alpha-ketoglutarate were identified in the intracellular pool of glutamine-incubated spheroplasts. The amino acids and peptides were incorporated into cell envelope material, and a portion (30 to 50%) of the incorporated amino acids could be removed by trypsinization or treatment with papain. The effect of glutamine was depressed by inhibitors of energy metabolism, suggesting that glutamine-stimulated incorporation is an energy-mediated effect.     

A single amino acid determinant of the membrane localization of lipoproteins in E. coli

When beta-lactamase was fused with the signal peptide plus the amino-terminal 9 amino acid residues of the major outer membrane lipoprotein, the resultant lipo-beta-lactamase (LL-1) was shown to be localized to the outer membrane. However, when the 9 residue sequence was replaced with the amino-terminal 12 residue sequence of lipoprotein-28, an inner membrane protein, the resultant lipo-beta-lactamase (LL-2) was found exclusively in the inner membrane. The localization of LL-2 was shifted to the outer membrane simply by substituting the second amino acid residue (Asp) of LL-2 with Ser. Conversely, the alteration of the second residue (Ser) of LL-1 to Asp resulted in the localization of LL-1 to the inner membrane. These results suggest that the second amino acid residue of the lipoproteins plays a crucial role in determining their final locations in the E. coli envelope.     

Pyridoxyl amino acid esters in peptide synthesis

Pyridoxyl residue was suggested to be used as a multifunctional protective and modifying group in peptide synthesis. The modification was carried out by introducing the pyridoxyl residue in free or partially protected peptides or by the addition of amino acid pyridoxyl esters by the methods of conventional peptide synthesis without the removal of the pyridoxyl group at the terminal stages of the synthesis (the second approach is more convenient). Pyridoxyl residue was also used as a spacer in solid phase peptide synthesis. It was attached to the polymer by the alkylation of the hydroxyl groups or of the pyridine ring of the pyridoxyl derivatives with the chloromethylated styrene-divinylbenzene copolymer (the standard Merrifield resin). Potentials for the use of pyridoxyl derivatives in the synthesis of linear, multiplet, and cyclic peptides are discussed.