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PubMed1992

Three widely separated positions in the 16S RNA lie in or close to the ribosomal decoding region; a site-directed cross-linking study with mRNA analogues.

Synthetic mRNA analogues were prepared by T7 transcription, each containing several thio-uridine residues at selected positions. After binding to the ribosome in the presence of cognate tRNA, the thio-U residues were activated by UV irradiation and the resulting sites of cross-linking to 16S RNA analysed. Three distinct cross-links were consistently observed: (i) from position '+6' of the mRNA (the 3'-base of the A-site codon) to base 1052 of 16S RNA; (ii) from position '+7' of the mRNA to base 1395; and (iii) from '+11' to base 532. Individual yields of the cross-links were strongly dependent on the particular mRNA sequence in each case. The '+11/532' and '+6/1052' cross-links were always entirely tRNA-dependent, whereas the '+7/1395' cross-link was observed at lower intensity in the absence of tRNA. In the presence of a second (A-site bound) tRNA the +6/1052 cross-link was markedly reduced. A cross-link to the 1050 region was again observed when a message carrying a thio-U at position '+9' was translocated on the ribosome so as to bring the thio-U to position +6. Taken together, the data are incompatible with some current models both for the three-dimensional arrangement of 16S RNA and for the orientation of the tRNA-mRNA complex in the ribosome.

Base Sequence↗
PubMed1992

Seven, eight and nine-membered anticodon loop mutants of tRNA(2Arg) which cause +1 frameshifting. Tolerance of DHU arm and other secondary mutations.

The mutant tRNA(2Arg) encoded by the genetically-selected frameshift suppressor, sufT621, inserts arginine and causes a +1 reading-frame shift at the proline codon, CCG(U). There is an extra base, G36.1, in argV beta, one of the four identical genes for tRNA(2Arg) in the position between bases 36 and 37, corresponding to the 3' side of the anticodon. The new four-base anticodon, predicted from DNA sequencing to be 3' GGCA 5', is complementary to the four-base codon CCGU. Quadruplet translocation promoted by mutant argV does not require perfect complementarity between the codon and the anticodon since synthetic genes encoding derivatives of tRNA(2Arg) and tRNA(1Pro), with four-base anticodons complementary to three out of the four bases of CCGU, were also shown to be capable of frameshifting. Two other mutants of argV, inferred to have normal-size, seven-base anticodon loops, were also found to be capable of four-base-decoding demonstrating that quadruplet translocation promoted by mutant argV does not require an enlarged anticodon loop. Other alleles of argV, predicted to have nine bases in the anticodon loop, were also found to cause frameshifting. The DNA sequence of two of these showed in addition, either a deletion of G24, or a ten-base duplication in the region corresponding to the TFC arm. A general finding is that mutations in the DHU arm of tRNA(2Arg) are compatible with, and in one case necessary for, frameshifting.

Amino Acid Sequence↗
PubMed1992

Mutational analysis of the "slippery-sequence" component of a coronavirus ribosomal frameshifting signal.

The ribosomal frameshift signal in the genomic RNA of the coronavirus IBV is composed of two elements, a heptanucleotide "slippery-sequence" and a downstream RNA pseudoknot. We have investigated the kinds of slippery sequence that can function at the IBV frameshift site by analysing the frameshifting properties of a series of slippery-sequence mutants. We firstly confirmed that the site of frameshifting in IBV was at the heptanucleotide stretch UUUAAAC, and then used our knowledge of the pseudoknot structure and a suitable reporter gene to prepare an expression construct that allowed both the magnitude and direction of ribosomal frameshifting to be determined for candidate slippery sequences. Our results show that in almost all of the sequences tested, frameshifting is strictly into the -1 reading frame. Monotonous runs of nucleotides, however, gave detectable levels of a -2/+1 frameshift product, and U stretches in particular gave significant levels (2% to 21%). Preliminary evidence suggests that the RNA pseudoknot may play a role in influencing frameshift direction. The spectrum of slip-sequences tested in this analysis included all those known or suspected to be utilized in vivo. Our results indicate that triplets of A, C, G and U are functional when decoded in the ribosomal P-site following slippage (XXXYYYN) although C triplets were the least effective. In the A-site (XXYYYYN), triplets of C and G were non-functional. The identity of the nucleotide at position 7 of the slippery sequence (XXXYYYN) was found to be a critical determinant of frameshift efficiency and we show that a hierarchy of frameshifting exists for A-site codons. These observations lead us to suggest that ribosomal frameshifting at a particular site is determined, at least in part, by the strength of the interaction of normal cellular tRNAs with the A-site codon and does not necessarily involve specialized "shifty" tRNAs.

Base Sequence↗
PubMed1987

Decoding at the ribosomal A site: antibiotics, misreading and energy of aminoacyl-tRNA binding.

The binding of Phe-tRNAPhe at the programmed ribosomal A site has been investigated using antibiotics that influence this binding in different ways. The adhesion of Phe-tRNAPhe, the consumption of GTP and the extent of the peptidyl transfer reaction were monitored. All of the five known misreading-inducing antibiotics that were tested stabilised the binding of Phe-tRNAPhe after its affixture to the A site by EF-Tu with GTP hydrolysis. The stabilisation was sufficient to overcome a single mismatch in the codon-anticodon interaction. Combinations of stabilising and destabilising influences were found to be additive, thus supporting the concepts: (1) that there is a 'correct' binding energy for aminoacyl tRNA in the A site, whose reduction hampers polypeptide synthesis and whose increase makes it inaccurate by by-passing proofreading; and (2) that the different antibiotics affect the bound aminoacyl tRNA at different points.

Anti-Bacterial Agents↗
PubMed1991

Selenocysteyl-tRNA occurs in the diatom Thalassiosira and in the ciliate Tetrahymena.

Selenocysteyl-tRNAs that decode UGA were identified previously in animal and bacterial cells and the genes for these tRNAs have been shown to be widespread in animals and eubacteria. In the present study, we identify a selenocysteyl-tRNA that codes for UGA in Thalassiosira pseudonana, which is a diatom, and in Tetrahymena borealis, which is a ciliate. The fact that these very diverse unicellular organisms also contain a selenocysteyl-tRNA suggests that selenocysteine-containing proteins and the use of UGA as a codon for selenocysteine are widespread, if not ubiquitous, in nature.

Animals↗
PubMed1988

Mutants of translational components that alter reading frame by two steps forward or one step back.

External suppressors, sufS, of a -1 frameshift mutant cause ribosomes to shift into the -1 frame when reading the sequence CAG GGA GUG. The resulting product is not Gln-Gly-Val but Gln-Gly-Ser with Ser being encoded by the underlined AGU. The alleles investigated are approximately 2% efficient in causing frameshifting. Two other suppressors, hopR and hopE of the same -1 frameshift mutant, cause some ribosomes reading the sequence GUG UG to decode a single amino acid, Val, from the five nucleotides. The possibility is considered that peptidyl-tRNA(Val) dissociates from the mRNA, but re-pairs in a triplet manner after the mRNA slips forward by two bases.

Amino Acid Sequence↗
PubMed1984

Crosslinking transfer RNA and messenger RNA at the ribosomal decoding region: identification of the site of reaction on the messenger RNA.

Wybutine (Ywye), situated next to the 3'-side of the anticodon of tRNAPhe from Saccharomyces cerevisiae, can be photo-crosslinked to mRNA when bound to Escherichia coli ribosomes. Crosslinking can be obtained with poly(U) as well as with oligonucleotides such as pAUGUUU or p(U)6. In order to identify the site of reaction on the mRNA, 5'-[32P]-labelled pAUGUUU was crosslinked by irradiation at 320 nm with Phe-tRNAPhe from yeast bound to the acceptor-site. The photoproduct was subsequently digested with P1-nuclease and analyzed by electrophoresis followed by homochromatography in the second dimension. As a result of the photoreaction the wybutine was found to be crosslinked to the U at the 5'-position of the corresponding UUU-codon.

Base Sequence↗
PubMed1986

Effects of post-transcriptional base modifications on the site-specific function of transfer RNA in eukaryote translation.

The site-specific function in translation of several naturally occurring mammalian transfer RNAs has been studied in a series of investigations with some similarities to studies in other laboratories of tRNAs in suppression. Equal amounts of aminoacyl-tRNA isoacceptors with contrasting isotopes were added in pairs to reticulocyte lysates and allowed to incorporate their amino acids into rabbit globin. Rates of incorporation from unlimiting amounts of each isoacceptor into the corresponding amino-acid-containing sites were determined. The tRNAs of each isoacceptor pair differed as to post-transcriptional base modifications. The natural occurrence of these isoacceptors can be correlated with rates of cellular division, with more rapidly dividing and neoplastic cells containing hypomodified tRNA. The overall incorporation of lysine into globin from a fully modified tRNALys that decodes AAG is faster by 25 to 30% than from the corresponding hypomodified tRNALys. There is considerable scatter in values for incorporation ratios at different lysine-containing sites, with the hypomodified isoacceptor even being preferred at one site. The AAG decoding isoacceptors are capable of translating AAA although much more slowly than AAG. In translating AAA, in contrast to translating AAG, the hypomodified tRNALys isoacceptor is preferred. A Y base-deficient hypomodified tRNAPhe isoacceptor found only in some kinds of rapidly dividing tumor cells donates its phenylalanine preferentially to globin in competition with the fully modified Y-containing tRNAPhe of liver by 15 to 17%. There is a considerable range of incorporation ratios at the different phenylalanine-containing sites of the globin subunits. No correlation can be made between the isoacceptor preferred and the phenylalanine codon being translated. The incorporation of histidine from a fully modified tRNAHis-containing Q base in its anticodon, compared with that from the hypomodified counterpart isoacceptor that lacks Q base and that occurs in rapidly dividing cells, showed no difference in their ability to incorporate overall or into individual histidine-containing sites. There is little evidence that adjacent bases or codons in messenger RNA affect the tRNAs preferred in the translation of most sites. A striking pattern of tRNA preference was observed in three cases in which there are tandem codons, with the same codon appearing twice in succession.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1985

Queuosine modification of the wobble base in tRNAHis influences 'in vivo' decoding properties.

The 'in vivo' decoding properties of four tRNAHis isoacceptors, two from Drosophila melanogaster and two from brewer's yeast, were studied after their microinjection, along with turnip yellow mosaic virus (TYMV) coat protein mRNA, into Xenopus laevis oocytes. The two Drosophila isoacceptors are identical besides containing either a guanosine (G) or the hypermodified nucleoside queuosine (Q) in the wobble position. The brewer's yeast isoacceptors differ by four bases in the anticodon stem, and by one base in the amino acceptor stem. Our results show that, under competing 'in vivo' conditions, the Drosophila tRNAHis with the anticodon GUG clearly prefers the histidine codon CAC to the codon CAU, whereas little preference is observed for the tRNAHis with the anticodon QUG for the codon CAU, and no preference for either codon by the two yeast isoacceptors. Hence, it can be concluded that the presence of the Q-base clearly affects the choice of the codon. This is the first demonstration of an 'in vivo' codon preference by tRNA isoacceptors differing in the modification of the wobble base during the elongation step of protein synthesis. These results imply that one function of the Q-base is at the translational level.

Animals↗
PubMed1984

High resolution localization of the tRNA anticodon interaction site on the Escherichia coli 30 S ribosomal subunit.

A body of previous work has shown that when Escherichia coli tRNAVal1 is placed in the P site of E. coli ribosomes and irradiated, the 5'-anticodon base of this tRNA, 5-carboxymethoxyuridine, is cross-linked to C-1400 of the 16 S rRNA. By tagging the carboxyl group of the cross-linked tRNA residue with a 2,4-dinitrophenyl (DNP) group attached via a 9 A spacer, it has been possible to directly visualize this cross-linking site by immunoelectron microscopy. The DNP group was attached by addition of ethylenediamine to the carboxyl group, followed by condensation of the newly formed free amino group with the N-hydroxysuccinimide ester of N-2,4-dinitrophenyl-gamma-aminobutyric acid. When reacted with anti-DNP antibody, this modification brings the surface of the antibody to within 9 A of the pyrimidine ring which was cross-linked. Neither codon-dependent binding nor cross-linking were materially affected by the tRNA modification. The tRNA-ribosome adduct formed a stable complex with anti-DNP antibody only when 50-30 S subunit association was prevented. Electron microscopic examination of the immune complexes showed that greater than 95% of those detected had the antibody localized deep in the cleft which separates the head and neck of the 30 S from the large protrusion. Since this is the site of cross-linking of the anticodon of tRNA, we conclude that this region on the 30 S subunit corresponds to the decoding site.

Antibodies↗
PubMed1990

Structure and expression of the Euglena gracilis nuclear gene coding for the translation elongation factor EF-1 alpha.

A cDNA library from the protist Euglena gracilis was used to isolate and sequence an ORF coding for the elongation factor protein EF-1 alpha. The decoded amino acid sequence (MW, 48'515) is to 75-80% identical with other eukaryotic EF-1 alpha sequences but only to 24% identical with the Euglena chloroplast EF-Tu. Homologous DNA probes interact with multiple fragments of Euglena nuclear restricted DNA typical for a multimembered gene family. We present the restriction sites map of four tef nuclear gene loci and postulate that the nuclear genome also contains tef related sequences (e.g. pseudogenes). Expression of tef gene(s) is monitored by Northern hybridization and the 5' end of a stable transcript (1.5 kb) is sequenced and shown to precede the start codon by 29 positions only. The steady state concentration of the 1.5 kb mRNA is not influenced by switching cell growth conditions from dark to light (chloroplast development).

Amino Acid Sequence↗
PubMed2025

Decoding the distribution, structure-function-redox potential relationship and recent advances in fungal laccases: a systematic approach.

Laccases, categorized as multicopper oxidases, are recognized for their multifaceted roles in ecosystems and their utility in diverse industrial applications. Laccases from higher fungi, specifically Ascomycota and Basidiomycota, have garnered significant research interest due to their elevated redox potentials and their capacity to degrade lignin in decaying wood, alongside other industrial uses. Here, we have conducted a comprehensive and systematic analysis on fungal laccases using Web of Science, Scopus, PubMed, and ScienceDirect. The genomic distribution, phylogenetic affiliation, and structural organization of laccase-encoding genes in higher fungal species were investigated, as were the catalytic mechanisms of the corresponding enzymes. Additionally, the study explores the correlation between structural domains and redox potential, as well as the impact of post-translational modifications like glycosylation on enzyme activity. Furthermore, the recent advancements in laccase engineering, employing strategies such as rational design, directed evolution, and heterologous expression are discussed. The review also explores the scope of "artificial intelligence and machine learning" in deducing the structure-function relationships, optimizing codon usage, predicting signal peptides, enhancing enzymatic performance, and developing host-specific genetic engineering techniques is also discussed for tailoring fungal laccases to meet the demands of industrial biocatalysis for improved activity and stability.

Laccase↗
PubMed1990

Significance of the third tRNA binding site, the E site, on E. coli ribosomes for the accuracy of translation: an occupied E site prevents the binding of non-cognate aminoacyl-tRNA to the A site.

The E site (exit site for deacyl-tRNA) has been shown to be allosterically linked to the A site (aminoacyl-tRNA binding site), in that occupation of the E site reduces the affinity of the A site, and vice versa, whereas the intervening peptidyl-tRNA binding site (P site) keeps its high affinity. Here the question is analysed of whether or not the low affinity state of the A site caused by an occupied E site is of importance for the ribosomal accuracy of the aminoacyl-tRNA selection. In a poly(U) dependent system with high accuracy in poly(Phe) synthesis, the acceptance of the cognate ternary complex Phe-tRNA--EF-Tu--GTP (which has the correct anticodon with respect to the codon at the A site) was compared with the competing acceptance of ternary complexes with near-cognate Leu-tRNA(Leu) (which has a similar anticodon) or non-cognate Asp-tRNA(Asp) (which has a dissimilar anticodon), by monitoring the formation of AcPhePhe, AcPheLeu or AcPheAsp, respectively. Cognate (but not near-cognate) occupation of the E site reduced synthesis of the 'wrong' dipeptide AcPheLeu only marginally relative to that of the cognate AcPhe2, whereas the formation of AcPheAsp was decreased as much as 14-fold, thereby reducing it to the background level. It follows that the allosteric interplay between E and A sites, i.e. the low affinity of the A site induced by the occupation of the E site, excludes the interference of non-cognate complexes in the decoding process and thus reduces the number of aminoacyl-tRNA species competing for A site binding by an order of magnitude.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1987

Reading frame selection and transfer RNA anticodon loop stacking.

Messenger RNA's are translated in successive three-nucleotide steps (a reading frame), therefore decoding must proceed in only one of three possible frames. A molecular model for correct propagation of the frame is presented based on (i) the measured translational properties of transfer RNA's (tRNA's) that contain an extra nucleotide in the anticodon loop and (ii) a straightforward concept about anticodon loop structure. The model explains the high accuracy of reading frame maintenance by normal tRNA's, as well as activities of all characterized frameshift suppressor tRNA's that have altered anticodon loops.

Anticodon↗
PubMed1992

Selenocysteyl-tRNAs recognize UGA in Beta vulgaris, a higher plant, and in Gliocladium virens, a filamentous fungus.

Selenocysteyl-tRNAs that decode UGA were previously identified in representatives of three of the five life kingdoms which were the monera, animal and protist kingdoms. In the present study, we show that these tRNAs also occur in representatives of the two remaining kingdoms, plants and fungi; i.e., selenocysteyl-tRNAs which code for UGA occur in Beta vulgaris, a higher plant, and in Gliocladium virens, a filamentous fungus. The fact that selenocysteyl-tRNAs are present in all five life kingdoms strongly suggests that UGA, in addition to dictating the cessation of protein synthesis, also codes for selenocysteine in the universal genetic code.

Base Sequence↗
PubMed1989

In vitro assembly of 30S and 70S bacterial ribosomes from 16S RNA containing single base substitutions, insertions, and deletions around the decoding site (C1400).

An in vitro system developed for the site-specific mutagenesis of 16S RNA of Escherichia coli ribosomes [Krzyzosiak et al. (1987) Biochemistry 26, 2353-2364] was used to make 10 single base changes around C1400, a residue known to be at the decoding site. C1400 was replaced by U, A, or G, five single base deletions at and to either side of C1400 were made, and C or U was inserted next to C1400. Another mutant possessed seven additional nucleotides at the 3' end of the 16S RNA such that a stem and loop involving the anti-Shine-Dalgarno sequence could form. Each of the mutant RNAs was reconstituted with a complete mixture of 30S proteins to yield 30S ribosomes. Modified in vitro reconstitution conditions were required to obtain assembly of all of the synthetic ribosomes. Quantitative HPLC analysis of the protein content of each mutant showed that all of the proteins were present. The ability of synthetic 30S to form 70S particles under functional assay conditions was about 75% that of natural 30S and was unchanged by any of the mutations except for the deletion of G1401, which decreased the association activity under the standard conditions to 35-40% of synthetic 30S. That part of the ribosomal P site which interacts with the anticodon loop of tRNA was investigated by near-UV (greater than 300 nm) induced cross-linking of AcVal-tRNA. Cross-linking depended on both 30S subunits and the correct codon. The cross-linking yield of all mutants with a pyrimidine at position 1400 was equal to control isolated 30S, and the first-order rate constants for cross-linking of those mutants tested were like reconstituted natural 30S. The site of cross-linking for mutants with a C or U insertion between C1400 and G1401 was shifted to the inserted residue. Cross-linking to the base 5' to G1401 rather than to the residue 3' to C1399 indicates that G1401 is an important structural determinant of the P site.

Centrifugation, Density Gradient↗