Nonenzymic translocation and spontaneous release of noncognate peptidyl transfer ribonucleic acid from Escherichia coli ribosomes

Poly(uridylic acid)-programmed ribosomes have been used to synthesize the noncognate peptidyl-tRNA Ac-Phe-Tyr-tRNATyr and its cognate counterpart Ac(Phe)2-tRNAPhe. After synthesis, Ac(Phe)2-tRNAPhe remains, as expected, in the ribosomal acceptor (A) site, but the noncognate AcPhe-Tyr-tRNATyr does not; part of it spontaneously falls off the ribosome and the rest translocates, without elongation factor (EF) G, to the ribosomal donor site. The inhibitor of translocation viomycin prevents both the spontaneous release and the nonenzymatic translocation by confining the noncognate peptidyl-tRNA to the A site. Under these conditions, the interaction of AcPhe-Tyr-tRNATyr with the A site appears to be similar to that of Ac(Phe)2-tRNAPhe without the antibiotic, and EF-G promotes the translocation and subsequent elongation of both peptidyl-tRNAs to comparable extents. The results indicate that, without viomycin, the noncognate peptidyl-tRNA is weakly held in the ribosomal A site and support the proposal that the release of peptidyl-tRNA occurring during protein synthesis in vivo is related to a ribosomal editing mechanism which discards mistranslated nascent proteins [Menninger, J. R. (1977) Mech. Ageing Dev. 6, 131].     

70-S ribosomes of Escherichia coli have an additional site for deacylated tRNA binding

Escherichia coli 70-S ribosomes contain a third site for tRNA binding, additional to the A and P sites. This conclusion is based on several findings. Direct measurements showed that in the presence of poly(U), when both A and P sites are occupied by Ac[14C]Phe-tRNAPhe, ribosomes are capable of binding additionally deacylated non-cognate [3H]tRNA. If ribosomes in the preparation are active enough, the total binding of labeled ligands amounted to 2.5 mol/mol ribosomes. In the absence of poly(U), when the A site can not bind, the P site and the 'additional' site can be filled simultaneously with Ac[14C]Phe-tRNAPhe and deacylated [3H]tRNA, or with [3H]tRNA alone; the total binding exceeds in this case 1.5 mol/mol ribosomes. The binding at the 'additional' site is not sensitive to the template. [3H]tRNA bound there is able to exchange rapidly for unlabeled tRNA in solution. Deacylated tRNA is preferred to the aminoacylated one. The binding of AcPhe-tRNAPhe was not observed there at all. The 3'-end adenosine is essential for the affinity. The function of the 'additional' site is not known, but its existence has to be considered when tRNA . ribosome complexes are studied.     

Sexual transmission of transposed activator elements in transgenic tomatoes

The transmission of transposed Ac elements in progeny derived by self-pollination of ten transformed tomato plants has been examined by Southern hybridization analysis. We show that six of these primary transformants have transmitted a transposed Ac to at least one progeny. One of the families was segregating for at least two different insertion events. In five of ten families, progeny were detected that contained a transposed Ac but no donor T-DNA sequences, indicating that a recombination event occurred between the original and new Ac insertion site. Somatic transposition of Ac as late as the R2 generation is evidenced. One family contained an empty donor site fragment but Ac was not detected in either the parent or progeny, indicating Ac was lost in this population early in regeneration. While four of ten families were segregating for aberrant phenotypes, there was no evidence that the mutated gene was linked to a transposed Ac.     

Transposition of Ac from the P Locus of Maize into Unreplicated Chromosomal Sites

We have analyzed donor and target sites of the mobile element Activator (Ac) that are altered as a result of somatic transposition from the P locus in maize. Previous genetic analysis has indicated that the two mitotic daughter lineages which result from Ac transposition from P differ in their Ac constitution at the P locus. Both lineages, however, usually contain transposed Ac elements which map to the same genetic position. Using methylation-sensitive restriction enzymes and genomic blot analysis, we identified Ac elements at both the donor P locus and Ac target sites and used this assay to clone the P locus and to identify transposed Ac elements. Daughter lineages were shown to be mitotic descendants from a single transposition event. When both lineages contained Ac genetic activity, they both contained a transposed Ac element on identical genomic fragments independent of the genetic position of the target site. This indicates that in the majority of cases, Ac transposition takes place after replication of the donor locus but before completion of replication at the target site.     

The enzymatic binding of aminoacyl-tRNA to reticulocyte ribosomes: The stimulatory effect of donor site bound peptidyl-tRNA

The affinity of ribosomes for the elongation factors EF₁ and EF₂ changes while the ribosome is going through the different steps of the elongation cycle. In this communication we provide evidence that the affinity of the EF₁-aa-tRNA-GTP complex for the ribosomal acceptor site differs for ribosomes having their donor site either vacant or occupied by peptidyl-tRNA or by uncharged tRNA. Ribosomes having peptidyl-tRNA at their donor site bind the EF₁ complex with the highest affinity.Results are discussed in light of recent findings that the two elongation factors are not bound to the ribosome simultaneously.     

Photoaffinity labeling of the ribosomal peptidyl transferase site with synthetic puromycin analogues.

A photoaffinity labeling puromycin analogue, Nepsilon-(2-nitro-4-azidophenyl)-L-lysinyl puromycin aminonucleoside (NAP-Lys-Pan), was synthesized and used for investigation of the peptidyl transferase center of 70S riobsomes. Visible light irradiation of NAP-Lys-Pan led to covalent linkage of the analogue with Escherichia coli ribosomes. In a subsequent step, poly(uridylic acid) was employed to direct Ac[14C]Phe-tRNA to the P sites of the photolabeled ribosomes. Transpeptidation of Ac[14C]phenylalanine to the bound NAP-Lys-Pan resulted in selective incorporation of radioactive label into the peptidyl transferase A site. Dissociation of the ribosomes into subunits, and digestion of the RNA components, indicated that the radioactive label was incorporated into a protein fraction of the 50S subunit.     

Distribution of Unlinked Receptor Sites for Transposed Ac Elements from the Bz-M2(ac) Allele in Maize

We have shown before that the Ac element from the maize bz-m2(Ac) allele, located in the short arm of chromosome 9 (9S), transposes preferentially to sites that are linked to the bz donor locus. Yet, about half of the Ac transpositions recovered from bz-m2(Ac) are in receptor sites not linked to the donor locus. In this study, we have analyzed the distribution of those unlinked receptor sites. Thirty-seven transposed Ac (trAc) elements that recombined independently of the bz locus were mapped using a set of wx reciprocal translocations. We found that the distribution of unlinked receptor sites for trAs was not random. Ten trAcs mapped to 9L, i.e., Ac had transposed to sites physically, if not genetically, linked to the donor site. Among chromosomes other than 9, the Ac element of bz-m2(Ac) appeared to have transposed preferentially to certain chromosomes, such as 5 and 7, but infrequently to others, such as 1, the longest chromosome in the maize genome. The seven trAc elements in chromosome 5 were mapped relative to markers in 5S and 5L and localized to both arms of 5. We also investigated the transposition of Ac to the homolog of the donor chromosome. We found that Ac rarely transposes from bz-m2(Ac) to the homologous chromosome 9. The clustering of Ac receptor sites around the donor locus has been taken to mean that a physical association between the donor site and nearby receptor sites occurs during transposition. The preferential occurrence of 9L among chromosomes harboring unlinked receptor sites would be expected according to this model, since sites in 9L would tend to be physically closer to 9S than sites in other chromosomes. The nonrandom pattern seen among the remaining chromosomes could reflect an underlying nuclear architecture, i.e., an ordering of the chromosomes in the interphase nucleus, as suggested from previous cytological observations.     

Interaction of substrates and substrate-like inhibitors with the peptidyltransferase center from Escherichia coli ribosomes

The binding isotherms of CACCA(3'NHPhe----Ac) and CACCA(3'NHPhe) to E. coli ribosomes and 50S subunits were measured. A theoretical model of adsorption for the case of cooperative interaction between two ligands adsorbed on a ribosome was designated. The analysis of the experimental binding isoterms leads to the following conclusions. A ribosome (or subunit) binds one CACCA (3'NHPhe----Ac) molecule to donor site of the peptidyl transferase center, but two CACCA (3'NHPhe) molecules to both donor and acceptor sites. The binding of CACCA (3'NHPhe) to ribosomes (or subunits) is a cooperative process, characterized by the cooperativity coefficient tau = 40 +/- 5 or more. When model substrates CACCA-Phe, CACCA-Leu and CACCA-Val were taken instead of CACCA (3'NHPhe) in the incubation mixture with ribosomes, dipeptides were obtained even in the case, when ratio [model substrate]: [ribosome] (in moles) was much lower than 1. Puromycin binding to acceptor site with constant (1-2) X 10(4) M-1 also stimulates CACCA(3'NHPhe----Ac) adsorption to the donor site of ribosomes with cooperativity coefficient being equal to 1.5-2.5. It is also shown that cytidine 5'-phosphate binding to the donor site increases kappa cat of the reaction of minimal donors with CACCA-Phe by 1.5 orders of magnitude but has no effect on Km of this reaction. These facts point out that cytidine 5'-phosphate being adsorbed on the corresponding area of the donor site leads to the conversion of low-productive complex [ribosome + minimal donor substrate + acceptor substrate] into high-productive complex [ribosome + minimal donor substrate + acceptor substrate + cytidine 5'-phosphate].     

Analysis of the puromycin reaction. The ribosomal exclusion principle for AcPhe-tRNA binding re-examined

The standard technique for determination of the ribosomal site location of bound tRNA, viz. the puromycin reaction, has been analyzed with regard to its applicability under tRNA saturation conditions. The criteria derived have been used to re-examine the exclusion principle for peptidyl-tRNA binding, which states that only one peptidyl-tRNA (AcPhe-tRNA) can be bound per ribosome although in principle two sites (A and P site) are available. The following results were obtained. The puromycin reaction is only appropriate for a site determination if the reaction conditions prevent one ribosome from performing more than one puromycin reaction. With an excess of AcPhe-tRNA over ribosomes, and in the absence of EF-G, this criterion is fulfilled at 0 degree C, where the P-site-bound material reacts with puromycin (quantitative reaction after 50 h), while the A-site-bound material does not. In contrast, at 37 degrees C the extent of the puromycin reaction can exceed the binding values by 2-4-fold ('repetitive reaction'). In the presence of EF-G a repetitive puromycin reaction is seen even at 0 degree C, i.e. EF-G can already promote a translocation reaction at 0 degree C. However, the extent of translocation becomes negligibly low for short incubation times (up to 60 min) at 0 degree C, if only catalytic amounts of EF-G are used. Using the criteria outlined above, the validity of the exclusion principle for Escherichia coli ribosomes was confirmed pursuing two different experimental strategies. Ribosomes were saturated with AcPhe-tRNA at one molecule per 70S ribosome, and a quantitative puromycin reaction demonstrated the exclusive P-site location of the AcPhe-tRNA. The same result was also found in the presence of viomycin, which blocks the translocation reaction. These findings also indicate that here nearly 100% of the ribosomes participate in AcPhe-tRNA binding to the P site. Precharging the P sites of 70S ribosomes with one Ac[14C]Phe-tRNA molecule per ribosome prevented additional Ac[3H]Phe-tRNA binding. In contrast, 70S particles carrying one molecule of [14C]tRNAPhe per ribosome were able to bind up to a further 0.64 molecule Ac[3H]Phe-tRNA per ribosome.     

Transposition Pattern of the Maize Element Ac from the Bz-M2(ac) Allele

The pattern of transposition of Ac from the mutable allele bz-m2(Ac) has been investigated. Stable (bz-s) and finely spotted (bz-m(F)) exceptions were selected from coarsely spotted bz-m2(Ac) ears. The presence or absence of a transposed Ac (trAc) in the genome was determined and, when present, the location of the trAc was mapped relative to the flanking markers sh and wx. The salient general features of Ac transposition to sites linked to bz are that the receptor sites tend to be clustered on either side of the bz donor site and that transposition is bidirectional and nonpolar. Thus, the symmetrical clustering in the distribution of receptor sites adjacent to bz differs from the asymmetrical clustering reported in 1984 for the P locus by I. M. GREENBLATT. Though Ac tends to transpose preferentially to closely linked sites, an appreciable fraction of Ac transpositions from bz-m2(Ac) is to unlinked sites: 41% among bz-s derivatives and 59% among bz-m(F) derivatives. Many transposition events among the bz-m(F) selections result in kernels carrying a genetically noncorresponding embryo. These can be interpreted as twin sectors arising at one of the megagametophytic mitoses. The bz locus data fit the earlier (1962) model of I. M. GREENBLATT and R. A. BRINK in which transposition takes place from a replicated donor site to either an unreplicated or replicated receptor site.     

Codon-anticodon interaction at the ribosomal E site

The question of whether or not the tRNA at the third ribosomal binding site specific for deacylated tRNA (E site) undergoes codon-anticodon interaction was analyzed as follows. Poly(U)-programmed ribosomes each carrying two [14C]tRNAPhe molecules were subjected to a chasing experiment using various tRNA species. At 0 degree C Ac[3H]Phe-tRNAPhe did not trigger any chasing whereas deacylated cognate tRNAPhe provoked a strong effect; non-cognate tRNALys was totally ineffective. This indicates that the second [14C]tRNAPhe cannot be present at the A site but rather at the E site (confirming previous observations). In the presence of poly(U) or poly(A) ribosomes bound the cognate tRNA practically exclusively as second deacylated tRNA, i.e. [14C]tRNAPhe and [14C]tRNALys, respectively. Thus, the second deacylated tRNA binds in a codon-dependent manner. [14C]tRNALys at the P site and Ac[3H]Lys-tRNALys at the A site of poly(A)-primed ribosomes were translocated to the E and P sites, respectively, by means of elongation factor G. The E site-bound [14C]tRNALys could be significantly chased by cognate tRNALys but not by non-cognate tRNAPhe, indicating the coded nature of the E site binding. Additional evidence is presented that the ribosome accommodates two adjacent codon-anticodon interactions at either A and P or P and E sites.     

Partial release of AcPhe-Phe-tRNA from ribosomes during poly(U)-dependent poly(Phe) synthesis and the effects of chloramphenicol

Poly(U)-programmed 70S ribosomes can be shown to be 80% to 100% active in binding the peptidyl-tRNA analogue AcPhe-tRNA to their A or P sites, respectively. Despite this fact, only a fraction of such ribosomes primed with AcPhe-tRNA participate in poly(U)-directed poly(Phe) synthesis (up to 65%) at 14 mM Mg2+ and 160 mM NH4+. Here it is demonstrated that the apparently 'inactive' ribosomes (greater than or equal to 35%) are able to participate in peptide-bond formation, but lose their nascent peptidyl-tRNA at the stage of Ac(Phe)n-tRNA, with n greater than or equal to 2. The relative loss of early peptidyl-tRNAs is largely independent of the degree of initial saturation with AcPhe-tRNA and is observed in a poly(A) system as well. This observation resolves a current controversy concerning the active fraction of ribosomes. The loss of Ac(Phe)n-tRNA is reduced but still significant if more physiological conditions for Ac(Phe)n synthesis are applied (3 mM Mg2+, 150 mM NH4+, 2 mM spermidine, 0.05 mM spermine). Chloramphenicol (0.1 mM) blocks the puromycin reaction with AcPhe-tRNA as expected but, surprisingly, does not affect the puromycin reaction with Ac(Phe)2-tRNA nor peptide bond formation between AcPhe-tRNA and Phe-tRNA. The drug facilitates the release of Ac(Phe)2-4-tRNA from ribosomes at 14 mM Mg2+ while it hardly affects the overall synthesis of poly(Phe) or poly(Lys).     

Dual actions of viomycin on the ribosomal functions.

Viomycin was observed to inhibit poly[U]- or f2 RNA-directed protein synthesis in an $\text{E. coli}$ cell-free system. The former was more profoundly affected than the latter. Both initiation complex formation on the 30S ribosomal subunit and on 70S ribosomes were prevented by the antibiotic. In the peptide chain elongation process, viomycin did not significantly affect aminoacyl-tRNA binding to ribosomes and the peptidyl transferase reaction, but markedly inhibit translocation of peptidyl-tRNA from the acceptor site to the donor site. The mechanism of action of the drug appeared to be unique.     

The action of virginiamycin M on the acceptor, donor, and catalytic sites of peptidyltransferase

Virginiamycin M inhibits both peptide bond formation and binding of aminoacyl-tRNA to bacterial ribosomes, and induces a lasting inactivation of the 50 S subunit (50 S). In the present work, the effects of this antibiotic on the acceptor and donor sites of peptidyltransferase have been explored, in the presence of virginiamycin M as well as after its removal. Virginiamycin M inhibited the binding of puromycin to ribosomes and reduced both the enzymatic and nonenzymatic binding of Phe-tRNA to the A site by inducing its release from the ribosomes (similar effects were observed with 50 S), whereas the antibiotic had no effect on the binding of unacylated tRNAPhe to the same site. Moreover, virginiamycin M caused Ac-Phe-tRNA or Phe-tRNA to be released from the ribosomal P site, when complexes were incubated with unacylated tRNA, elongation factor G, and GTP (similar finding with 50 S). Instead, peptide bond formation between Ac-Phe-tRNA positioned at the P site and Phe-tRNA at the A site was found to take place, albeit at a very low rate, in the presence of the antibiotic. The overall conclusion is that both the acceptor and donor substrate binding sites of the peptidyltransferase, which interact with the aminoacyl moiety of tRNA, are permanently altered upon transient contact of ribosomes with virginiamycin M.     

Origination of Ds elements from Ac elements in maize: evidence for rare repair synthesis at the site of Ac excision.

Although it has been known for some time that the maize transposon Ac can mutate to Ds by undergoing internal deletions, the mechanism by which these mutations arise has remained conjectural. To gain further insight into this mechanism in maize we have studied a series of Ds elements that originated de novo from Ac elements at known locations in the genome. We present evidence that new, internally deleted Ds elements can arise at the Ac donor site when Ac transposes to another site in the genome. However, internal deletions are rare relative to Ac excision footprints, the predominant products of Ac transposition. We have characterized the deletion junctions in five new Ds elements. Short direct repeats of variable length occur adjacent to the deletion junction in three of the five Ds derivatives. In the remaining two, extra sequences or filler DNA is inserted at the junction. The filler DNAs are identical to sequences found close to the junction in the Ac DNA, where they are flanked by the same sequences that flank the filler DNA in the deletion. These findings are explained most simply by a mechanism involving error-prone DNA replication as an occasional alternative to end-joining in the repair of Ac-generated double-strand breaks.     

Crystal structures of Pasteurella multocida sialyltransferase complexes with acceptor and donor analogues reveal substrate binding sites and catalytic mechanism

Sialyltransferases are key enzymes involved in the biosynthesis of biologically and pathologically important sialic acid-containing molecules in nature. Binary X-ray crystal structures of a multifunctional Pasteurella multocida sialyltransferase (Delta24PmST1) with a donor analogue CMP-3F(a)Neu5Ac or CMP-3F(e)Neu5Ac were determined at 2.0 and 1.9 A resolutions, respectively. Ternary X-ray structures of the protein in complex with CMP or a donor analogue CMP-3F(a)Neu5Ac and an acceptor lactose have been determined at 2.0 and 2.27 A resolutions, respectively. This represents the first sialyltransferase structure and the first GT-B-type glycosyltransferase structure that is bound to both a donor analogue and an acceptor simultaneously. The four structures presented here reveal that binding of the nucleotide-activated donor sugar causes a buried tryptophan to flip out of the protein core to interact with the donor sugar and helps define the acceptor sugar binding site. Additionally, key amino acid residues involved in the catalysis have been identified. Structural and kinetic data support a direct displacement mechanism involving an oxocarbenium ion-like transition state assisted with Asp141 serving as a general base to activate the acceptor hydroxyl group.     

Transposition of reversed Ac element ends generates chromosome rearrangements in maize

In classical "cut-and-paste" transposition, transposons are excised from donor sites and inserted at new locations. We have identified an alternative pathway in which transposition involves the 5' end of an intact Ac element and the 3' end of a nearby terminally deleted fAc (fractured Ac). The Ac and fAc elements are inserted at the maize p1 locus on chromosome 1s in the same orientation; the adjacent ends of the separate elements are thus in reversed orientation with respect to each other and are separated by a distance of approximately 13 kb. Transposition involving the two ends in reversed orientation generates inversions, deletions, and a novel type of local rearrangement. The rearrangement breakpoints are bounded by the characteristic footprint or target site duplications typical of Ac transposition reactions. These results demonstrate a new intramolecular transposition mechanism by which transposons can greatly impact genome evolution.     

Insertional Mutagenesis of the Maize P Gene by Intragenic Transposition of Ac

The P-rr allele of the maize P gene regulates the synthesis of pigments derived from flavan-4-ol in the pericarp, cob glumes and other floral organs. We characterized 21 P alleles derived by intragenic transposition of Ac from three known positions. Ac transpositions can occur in either direction in the P gene, and with no apparent minimum distance: in one case Ac transposed just 6 bp from its original insertion site. However, the distribution of transposed Ac elements was markedly nonrandom: of 19 transposed Ac elements derived from a single Ac donor, 15 were inserted in a 1.1-kb region at the 5' end of P, while none had inserted in an adjacent 3.2-kb intronic region. All of the Ac insertions affect both pericarp and cob glume pigmentation, providing further evidence that the P-rr allele contains a single gene required for both pericarp and cob glume pigmentation. The distribution of the inserted Ac elements and the phenotype conditioned by each allele suggests a structure of P-rr which is similar to that previously determined molecularly. Possible explanations for the nonrandom distribution of transposed Ac elements are discussed.     

Transposition of the maize activator element in transgenic rice plants.

Transposition of the maize Activator (Ac) element was observed in transgenic rice. After protoplast transformation, Ac excision from an interrupted hygromycin phosphotransferase gene was monitored by appearance of the hygromycin-resistant colonies. The frequency of Ac excision, based on the biological assay was up to 19%. Southern hybridization analysis indicated that at least one copy per genome of the hygromycin-resistance gene was reconstituted after Ac excision and that the transposed Ac element was reintegrated into the rice genome. Analysis of DNA sequences at 14 empty donor sites indicated that the Ac element was excised in rice in a similar manner as maize. The excision of an Ac mutant in which a 1.3 kbp Tn903 fragment was inserted at a unique BamHI site so as to disrupt binding of the putative transposase was not detected by DNA analysis. These results demonstrated that the maize Ac element might be used as an effective heterologous transposon for mutagenesis and gene tagging in rice, an important food crops.     

Reconstitutional Mutagenesis of the Maize P Gene by Short-Range Ac Transpositions

The tendency for Ac to transpose over short intervals has been utilized to develop insertional mutagenesis and fine structure genetic mapping strategies in maize. We recovered excisions of Ac from the P gene and insertions into nearby chromosomal sites. These closely linked Ac elements reinserted into the P gene, reconstituting over 250 unstable variegated alleles. Reconstituted alleles condition a variety of variegation patterns that reflect the position and orientation of Ac within the P gene. Molecular mapping and DNA sequence analyses have shown that reinsertion sites are dispersed throughout a 12.3-kb chromosomal region in the promoter, exons and introns of the P gene, but in some regions insertions sites were clustered in a nonrandom fashion. Transposition profiles and target site sequence data obtained from these studies have revealed several features of Ac transposition including its preference for certain target sites. These results clearly demonstrate the tendency of Ac to transpose to nearby sites in both proximal and distal directions from the donor site. With minor modifications, reconstitutional mutagenesis should be applicable to many Ac-induced mutations in maize and in other plant species and can possibly be extended to other eukaryotic transposon systems as well.