Elongator tRNA Research Articles

Important Role of the Amino Acid Attached to tRNA in Formylation and in Initiation of Protein Synthesis in Escherichia coli

In attempts to convert an elongator tRNA to an initiator tRNA, we previously generated a mutant elongator methionine tRNA carrying an anticodon sequence change from CAU to CUA along with the two features important for activity of Escherichia coli initiator tRNA in initiation. This mutant tRNA (Mi:2 tRNA) was active in initiation in vivo but only when aminoacylated with methionine by overproduction of methionyl-tRNA synthetase. Here we show that the Mi:2 tRNA is normally aminoacylated in vivo with lysine and that the tRNA aminoacylated with lysine is a very poor substrate for formylation compared with the same tRNA aminoacylated with methionine. By introducing further changes at base pairs 4:69 and 5:68 in the acceptor stem of the Mi:2 tRNA to those found in the E. coli initiator tRNA, we show that change of the U4:A69 base pair to G4:C69 and overproduction of lysyl-tRNA synthetase and methionyl-tRNA transformylase results in partial formylation of the mutant tRNA and activity of the formyllysyl-tRNAs in initiation of protein synthesis. Thus, the G4: C69 base pair contributes toward formylation of the tRNA and protein synthesis in E. coli can be initiated with formyllysine. We also discuss the implications of these and other results on recognition of tRNAs by E. coli lysyl-tRNA synthetase and on competition in cells among aminoacyl-tRNA synthetases.

Specific RNA cleavages induced by manganese ions

The specificity and efficiency of manganese ion-induced RNA hydrolysis was studied with several tRNA molecules. In case of yeast tRNA Phe, the main cleavage occurs at p16 and minor cuts at p17–18, p20–21, p34 and p36–37. The major Mn(II)-induced cut in yeast elongator tRNA Met is also located in the D-loop at p16 and it is stronger than that observed in tRNA Phe. In initiator tRNA Met from yeast two strong Mn(II) cleavages of equal intensity occur at p16 and p17. This is in contrast with single, much weaker cuts induced in the D-loop of that tRNA by Mg(II), Eu(III) and Pb(II) ions. Interestingly, in case of yeast tRNA Glu the main cleavage caused by Mn(II), Mg(II) and Pb(II) ions occurs in the anticodon loop. The involvement of hypermodified base mnm 5s 2U in this cleavage was ruled out based on results obtained with in vitro transcript of yeast tRNA Glu anticodon arm. Mutation of a single base A37G in the anticodon loop of the transcript drastically reduced the specificity of Mn(II)-induced hydrolysis.

Nucleotide sequence of the psbE, psbF and trnM genes from the chloroplast genome of Chlamydomonas reinhardtii

We have determined the nucleotide sequences of the psbE and psbF genes, which encode the α and β subunits, respectively, of cytochrome b-559, from the chloroplast genome of the green alga Chlamydomonas reinhardtii. In contrast to other organisms psbE is not co-transcribed with psbF. The primary structures of the gene products are very similar to the equivalent proteins in cyanobacteria and plants. Each subunit contains a single histidine residue that is thought to ligate haem. Upstream of the psbE gene, a trnM gene is located which encodes an elongator tRNA Met molecule.

Rit1, a tRNA backbone-modifying enzyme that mediates initiator and elongator tRNA discrimination

Using a genetic screen in yeast aimed at identifying cellular factors involved in initiator and elongator methionine tRNA discrimination in the translational process, we have identified a mutation that abolish the requirement for elongator methionine tRNA. The gene affected, which we call the ribosylation of the initiator tRNA gene or RIT1, encodes a 2′-O-ribosyl phosphate transferase. This enzyme modifies exclusively the initiator tRNA in position 64 using 5′-phosphoribosyl-1′-pyrophosphate as the modification donor. As the initiator tRNA participates both in the initiation and elongation of translation in a rit1 strain, we conclude that the 2′-O-ribosyl phosphate modification discriminates the initiator tRNAs from the elongator tRNAs during protein synthesis. The modification enzyme was shown to recognize the stem-loop IV region that is unique in eukaryotic cytoplasmic initiator tRNAs.

Peptide-chain elongation in eukaryotes

The elongation phase of translation leads to the decoding of the mRNA and the synthesis of the corresponding polypeptide chain. In most eukaryotes, two distinct protein elongation factors (eEF-1 and eEF-2) are required for elongation. Each is active as a complex with GTP. eEF-1 is a multimer and mediates the binding of the cognate aminoacyl-tRNA to the ribosome, while eEF-2, a monomer, catalyses the movement of the ribosome relative to the mRNA. Recent work showing that bacterial ribosomes possess three sites for tRNA binding and that during elongation tRNAs may occupy 'hybrid' sites is incorporated into a model of eukaryotic elongation. In fungi, elongation also requires a third factor, eEF-3. A number of mechanisms exist to promote the accuracy or 'fidelity' of elongation: eEF-3 may play a role here. cDNAs for this and the other elongation factors have been cloned and sequenced, and the structural and functional properties of the elongation factors are discussed. eEF-1 and eEF-2 can be regulated by phosphorylation, and this may serve to control rates of elongation in vivo.

Analysis of acceptor stem base pairing on tRNA(Trp) aminoacylation and function in vivo.

The role of acceptor stem base pairs in determining the identity of Escherichia coli tRNA(Trp) was examined by complementation of an E. coli strain containing a temperature-sensitive tRNA(Trp) gene (trpTts) and by monitoring aminoacylation levels in vivo. All derivatives of tRNA(Trp) containing substitutions at the first 3 base pairs in the acceptor stem complemented the trpTts mutation at the nonpermissive temperature (42 degrees C). However, three acceptor stem derivatives (tRNA(Trp)/C1.G72, tRNA(Trp)/C2.G71, and tRNA(Trp)/A3.U70) required overexpression for growth at 42 degrees C. Northern analysis of these derivatives following acid/urea gel electrophoresis showed no defects in tRNA aminoacylation at the nonpermissive temperature. Instead, these tRNAs appear to be defective in translation. This was suggested by the weak opal suppressor activities of the corresponding tRNA(UCATrp) derivatives. These results demonstrate that the three terminal acceptor stem base pairs do not contribute to the identity of tRNA(Trp). Substitution of the C1.A72 base pair in a methionine initiator tRNA containing the tryptophan anticodon and discriminator base (tRNA(CCAfMet)/G73) with A1.U72, the base pair found in tRNA(Trp), or G1.C72 resulted in the conversion of these tRNAs into tryptophan-inserting elongator tRNAs in vivo. However, changes to U1.A72 or C1.G72 in tRNA(CCAfMet)/G73 resulted in misaminoacylation and/or defects in translation. Our data indicate that the A1.U72 base pair is a context-dependent, negative identity element of tRNA(Trp).

A human DNA segment encompassing leucine and methionine tRNA pseudogenes localized on chromosome 6

A human genomic clone, designated LHtlm8, that strongly hybridized to a mammalian leucine tRNA IAG probe, was found to encompass a pair of tRNA pseudogenes that are transcribed in a homologous cell extract. A leucine tRNA AAG pseudogene ( TRLP1) is 2.1-kb upstream and of opposite polarity to a methionine elongator tRNA CAU pseudogene ( TRMEP1). TRLP1 has three nucleotide variations (97% identity) from its cognate leucine tRNA IAG, while TRMEP1 has a 78% identity with its cognate tRNA. Similar to a number of other eukaryotic tRNA pseudogenes, presumptive precursor tRNA transcripts are generated from the two pseudogenes in vitro, but possibly due to their aberrant and unstable secondary and tertiary structures, no detectable mature tRNA products are observed. The two tRNA pseudogenes are encompassed within a 9.6-kb EcoRI fragment that has been assigned to the chromosomal locus, 6pter-q13, by Southern blot hybridization of human-rodent somatic cell hybrid DNAs with probes derived from the cloned tRNA pseudogenes and flanking sequences. A 4.4-kb EcoRI fragment also harbored in clone LHtlm8 was mapped to human chromosome 11, suggesting that the two EcoRI fragments were inadvertantly ligated together during construction of the genomic library.

Translation Initiation Complex Formation with 30 S Ribosomal Particles Mutated at Conserved Positions in the 3′-Minor Domain of 16 S RNA

Escherichia coli 30 S ribosomal subunits containing in vitro (phage T7 RNA polymerase-generated) 16 S rRNA, both wild-type and mutant, were examined by toeprinting. These synthetic particles were used to compare the effects of the absence of base modification and of specific nucleotide substitutions in conserved sequence regions of the RNA on the assembly of mRNA, tRNAs and 30 S particles into a translational initiation complex. Initiation factor-3-dependent selection of tRNA Met f from a mixture of tRNA Met f and tRNA Phe occurred with all particles, although 20 times less initiation factor-3 was needed for the synthetic particles, including the mutants. Whereas isolated 30 S particles or those reconstituted with isolated RNA did not distinguish between tRNA Met f and tRNA Phe for ternary complex formation in the absence of initiation factor-3 (intrinsic selection ability), the synthetic particles preferred tRNA Met f. The difference between the natural and synthetic particles appears to be due to the absence of certain base modifications, but not m 2 6A, in the synthetic RNA. Synthetic particles containing the mutation U1512C, which converts the universal U·G pair to C·G enhanced both tRNA Met f binding and selectivity, although other mutations at that site, namely U1512G, G1523A and U1512C/C1524U, had no such effect. Mutants U1498G and G1401C/C1501G, both located in a highly conserved single-stranded region of the 3′-minor domain, also enhanced tRNA Met f selectivity, in this case by reducing complex formation with elongator tRNA. Complex formation between elongator tRNA and the G1401C/C1501G mutant was reduced to almost undetectable levels. The results also indicated that the association rate for initiation complex formation for G1401C/C1501G was considerably lower than for the wild-type sequence. This result had not been detected by standard tRNA-30 S binding assays. Overall, the data suggest that (some of) the 16 S rRNA base modifications as well as the tertiary structure around the decoding site act to desensitize the intrinsic selection ability of the ribosome for tRNA Met f.

The Yeast Initiator tRNA Met can Act as an Elongator tRNA MetIn Vivo

Saccharomyces cerevisiae uses two different methionine accepting tRNAs during protein synthesis. One, tRNA Met i, is used exclusively during the initiation of translation whereas the other, tRNA Met m, is used during the elongation of translation. To study the unique features of each methionine tRNA species, we constructed yeast strains with null alleles of the five elongator methionine tRNA ( EMT) genes and strains with null alleles of the four initiator methionine tRNA ( IMT) genes, respectively. Consequently, growth of these strains was dependent either on a tRNA Met m or a tRNA Met i, respectively, encoded from a plasmid-derived gene. For both null mutants, the plasmid carrying the wild-type gene can be selected against and exchanged for another plasmid derived EMT or IMT gene (wild-type or mutant). A high gene dosage of the wild-type IMT gene could restore growth to the elongator-depleted strain. However, wild-type EMT genes in a high gene dosage never restored growth of the initiator depleted strain. Thus, the elongator tRNA Met is much more restricted to participate in the initiation of translation than the initiator tRNA Met is restricted to participate in the elongation process. Using the two null mutants, we have identified tRNA Met m mutants, which show reduced elongator activity, and tRNA Met i mutants, with improved elongator activity in the elongator depleted strain. Also, tRNA Met m mutants that function as an initiator tRNA in the initiator depleted strain were identified. From this mutant analysis, we showed that the conserved U/rT at position 54 of the elongator tRNA Met is an important determinant for an elongator tRNA. The most important determinant for an initiator was shown to be the acceptor stem and especially the conserved A 1 · U 72 base-pair. Mutant tRNAs, with reduced activity in either process, were investigated for enhanced activity during overproduction of the α and β-subunits of the eukaryotic initiation factor 2 (eIF-2) or the eukaryotic elongation factor 1α (eEF-1α). The data suggest that the U/rT of the elongator at position 54 is important for eEF-1α recognition and that the acceptor stem of the initiator is important for eIF-2 recognition.

Importance of formylability and anticodon stem sequence to give a tRNA(Met) an initiator identity in Escherichia coli

In bacteria, the free amino group of the methionylated initiator tRNA is specifically modified by the addition of a formyl group. The functional relevance of such a formylation for the initiation of translation is not yet precisely understood. Advantage was taken here of the availability of the fmt gene, encoding the Escherichia coli Met-tRNA(fMet) formyltransferase, to measure the influence of variations in the level of formyltransferase activity on the involvement of various mutant tRNA(fMet) and tRNA(mMet) species in either initiation or elongation in vivo. The data obtained established that formylation plays a dual role, firstly, by dictating tRNA(fMet) to engage in the initiation of translation, and secondly, by preventing the misappropriation of this tRNA by the elongation apparatus. The importance of formylation in the initiator identity of tRNA(fMet) was further shown by the demonstration that elongator tRNA(fMet) may be used in initiation and no longer in elongation, provided that it is mutated into a formylatable species and is given the three G.C base pairs characteristic of the anticodon stem of initiator tRNAs.

From elongator tRNA to initiator tRNA.

We show that the two most important properties needed for a tRNA to function in initiation in Escherichia coli are its ability to be formylated and its ability to bind to the ribosomal P site. This conclusion is based on conversion of two different elongator tRNAs to ones that can act as initiators in E. coli. We transplanted the features unique to E. coli and eubacterial initiator tRNAs to E. coli elongator methionine tRNA (tRNA(Met)) along with an anticodon sequence change and analyzed their activities in initiation in E. coli. Introduction of a C1.A72 mismatch at the end of the acceptor stem of tRNA(Met), which generates the minimal features necessary for formylation, produces a tRNA with very low activity in initiation. Subsequent introduction of three consecutive G.C base pairs at the bottom of the anticodon stem, which is necessary for ribosomal P site binding, produces a tRNA with significant activity in initiation. Furthermore, introduction of the features necessary for formylation and for ribosomal P site binding into E. coli elongator glutamine tRNA produces a tRNA that initiates protein synthesis in E. coli.

The sequence of the gene encoding elongation factor Tu from Chlamydia trachomatis compared with those of other organisms

Nucleotide (nt) sequences encoding the elongation factor Tu(EF-Tu), tRNA The and tRNA Trp from Chlamydia trachomatis have been determined. The environment of the EF-Tu-encoding gene ( tuf), between two tRNA gene sequences, suggests that it is part of a tufB locus. The nt sequence and the deduced amino acid (aa) sequence were aligned with comparable sequences from other organisms and the resulting data bases were used to infer phylogenies. Phylogenetic trees based on aa sequences and nt sequences are similar, but not completely congruent with rRNA gene-based phylogenies. Both the nt and aa sequence trees concur on the early divergence of Thermotoga and Chlamydia from the bacterial root. The aa alignment highlights the presence of four unique Cys residues in the chlamydial sequence which are found at strictly conserved positions in other sequences. Further peculiarities of the chlamydial and eubacterial sequences have been mapped to the X-ray crystallographic structure of the protein.

Conversion of a methionine initiator tRNA into a tryptophan-inserting elongator tRNA in vivo.

The role of the anticodon and discriminator base in aminoacylation of tRNAs with tryptophan has been explored using a recently developed in vivo assay based on initiation of protein synthesis by mischarged mutants of the Escherichia coli initiator tRNA. Substitution of the methionine anticodon CAU with the tryptophan anticodon CCA caused tRNA(fMet) to be aminoacylated with both methionine and tryptophan in vivo, as determined by analysis of the amino acids inserted by the mutant tRNA at the translational start site of a reporter protein containing a tryptophan initiation codon. Conversion of the discriminator base of tRNA(CCA)fMet from A73 to G73, the base present in tRNA(Trp), eliminated the in vivo methionine acceptor activity of the tRNA and resulted in complete charging with tryptophan. Single base changes in the anticodon of tRNA(CCA)fMet containing G73 from CCA to UCA, GCA, CAA, and CCG (changes underlined) essentially abolished tryptophan insertion, showing that all three anticodon bases specify the tryptophan identity of the tRNA. The important role of G73 in tryptophan identity was confirmed using mutants of an opal suppressor derivative of tRNA(Trp). Substitution of G73 with A73, C73, or U73 resulted in a large loss of the ability of the tRNA to suppress an opal stop codon in a reporter protein. Base pair substitutions at the first three positions of the acceptor stem of the suppressor tRNA caused 2-12-fold reductions in the efficiency of suppression without loss of specificity for aminoacylation of the tRNA with tryptophan.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleotides of tRNA governing the specificity of Escherichia coli methionyl-tRNA fMet formyltransferase

In Escherichia coli, the free amino group of the aminoacyl moiety of methionyl-tRNA f Met is specifically modified by a transformylation reaction. To identify the nucleotides governing the recognition of the tRNA substrate by the formylase, initiator tRNA f Met was changed into an elongator tRNA with the help of an in vivo selection method. All the mutations isolated were in the tRNA acceptor arm, at positions 72 and 73. The major role of the acceptor arm was further established by the demonstration of the full formylability of a chimaeric tRNA Met containing the acceptor stem of tRNA f Met and the remaining of the structure of tRNA m Met. In addition, more than 30 variants of the genes encoding tRNA m Met or tRNA f Met have been constructed, the corresponding mutant tRNA products purified and the parameters of the formylation reaction measured. tRNA m Met became formylatable by the only change of the G1 · C72 base-pair into C1-A72. It was possible to render tRNA m Met as good a substrate as tRNA f Met for the formylase by the introduction of a limited number of additional changes in the acceptor stem. In conclusion, A73, G2 · C71, C3 · G70 and G4 · C69 are positive determinants for the specific processing of methionyl-tRNA f Met by the formylase while the occurrence of a G · C or C · G base-pair between positions 1 and 72 acts as a major negative determinant. This pattern appears to account fully for the specificity of the formylase and the lack of formylation of any aminoacylated tRNA, excepting the methionyl-tRNA f Met.

Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian and E. coli methionyl-tRNA synthetases.

The rates of the cross-aminoacylation reactions of tRNAs(Met) catalyzed by methionyl-tRNA synthetases from various organisms suggest the occurrence of two types of tRNA(Met)/methionyl-tRNA synthetase systems. In this study, the tRNA determinants recognized by mammalian or E. coli methionyl-tRNA synthetases, which are representative members of the two types, have been examined. Like its prokaryotic counterpart, the mammalian enzyme utilizes the anticodon of tRNA as main recognition element. However, the mammalian cytoplasmic elongator tRNA(Met) species is not recognized by the bacterial synthetase, and both the initiator and elongator E. coli tRNA(Met) behave as poor substrates of the mammalian cytoplasmic synthetase. Synthetic genes encoding variants of tRNAs(Met), including the elongator one from mammals, were expressed in E. coli. tRNAs(Met) recognized by a synthetase of a given type can be converted into a substrate of an enzyme of the other type by introducing one-base substitutions in the anticodon loop or stem. In particular, a reduction of the size of the anticodon loop of cytoplasmic mammalian elongator tRNA(Met) from 9 to 7 bases, through the creation of an additional Watson-Crick pair at the bottom of the anticodon stem, makes it a substrate of the prokaryotic enzyme and decreases its ability to be methionylated by the mammalian enzyme. Moreover, enlarging the size of the anticodon loop of E. coli tRNA(Metm) from 7 to 9 bases, by disrupting the base pair at the bottom of the anticodon stem, renders the resulting tRNA a good substrate of the mammalian enzyme, while strongly altering its reaction with the prokaryotic synthetase. Finally, E. coli tRNA(Metf) can be rendered a better substrate of the mammalian enzyme by changing its U33 into a C. This modification makes the sequence of the anticodon loop of tRNA(Metf) identical to that of cytoplasmic initiator tRNA(Met).

Purification and initial characterization of peptidyl-tRNA hydrolase from rabbit reticulocytes.

We have identified an activity in rabbit reticulocyte lysate as peptidyl-tRNA hydrolase, based upon its ability to hydrolyze native reticulocyte peptidyl-tRNA, isolated from polyribosomes, and N-acylaminoacyl-tRNA, and its inability to hydrolyze aminoacyl-tRNA, precisely the same substrate specificity previously reported for peptidyl-tRNA hydrolase from bacteria or yeast. The physiological role of the reticulocyte enzyme may be to hydrolyze and recycle peptidyl-tRNA that has dissociated prematurely from elongating ribosomes, as suggested for the bacterial and yeast enzymes, since reticulocyte peptidyl-tRNA hydrolase is completely incapable of hydrolyzing peptidyl-tRNA that is still bound to polyribosomes. We have purified reticulocyte peptidyl-tRNA hydrolase over 5,000-fold from the postribosomal supernatant with a yield of 14%. The purified product shows a 72-kDa band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis that has co-purified with enzyme activity and comprises about 90% of the total stained protein, strongly suggesting that the 72-kDa protein is the enzyme. Sucrose density gradient analysis indicates an apparent molecular mass for the native enzyme of 65 kDa, implying that it is a single polypeptide chain. The enzyme is almost completely inactive in the absence of a divalent cation: Mg2+ (1-2 mM) promotes activity best, Mn2+ is partly effective, and Ca2+ and spermidine are ineffective. The hydrolase shows a Km of 0.60 microM and Vmax of 7.1 nmol/min/mg with reticulocyte peptidyl-tRNA, a Km of 60 nM and Vmax of 14 nmol/min/mg with Escherichia coli fMet-tRNA(fMet), and a Km of 100 nM and Vmax of 2.2 nmol/min/mg with yeast N-acetyl-Phe-tRNA(Phe). The enzyme has a pH optimum of 7.0-7.25, it is inactivated by heat (60 degrees C for 5 min), and its activity is almost completely inhibited by pretreatment with N-ethylmaleimide or incubation with 20 mM phosphate. The fact that the enzyme hydrolyzes E. coli but not yeast or reticulocyte fMet-tRNA(fMet) may be explained, at least in part, by structural similarities between prokaryotic tRNA(fMet) and eukaryotic elongator tRNA that are not shared by eukaryotic tRNA(fMet).

Unusual ribosome binding properties of mRNA encoding bacteriophage lambda repressor.

The mRNA encoding repressor cI of phage lambda is the only known E. coli message which starts directly with the initiation AUG codon. The ability of in vitro synthesized cI mRNA fragments (150 or 400 nts) to form ternary initiation complexes has been studied using the toeprint method. In the presence of tRNA(Met)f, these fragments are capable of forming the ternary complexes at the 5'-terminal AUG codon not only with 30S subunits but also with undissociated 70S ribosomes (70S tight couples). In the latter case, no binding at other positions of cI mRNA can be detected at all. The starting region of cI mRNA has a single stranded conformation and is highly enriched in A-residues. This feature of cI mRNA RBS is suggested to be the main factor which allows cI mRNA to form the initiation complex with the ribosome. Unlike 30S subunits, the binding to 70S tight couples is not affected by any of the initiation factors, although it is as efficient as that to 30S subunits supplemented with the factors. 30S subunits prefer to associate with the internal RBSs of the preformed mRNA molecules, provided that they are not sequestered by the secondary structure. In contrast, 70S tight couples tend to avoid extra sequences upstream of the codon directed to the P site and occupy a position as close as possible to the 5'-end of the message. This has been found to be the case both for tRNA(Met)f and for elongator tRNA(Glu)2. The structural features of mRNA RBSs which influence their different binding for 30S subunits and 70S ribosomes are discussed.

A synthetic alanyl-initiator tRNA with initiator tRNA properties as determined by flourescence measurements: comparison to a synthetic alanyl-elongator tRNA

Two synthetic tRNAs have been generated that can be enzymatically aminoacylated with alanine and have AAA anticodons to recognize a poly(U) template. One of the tRNAs (tRNA(eAla/AAA)) is nearly identical to Escherichia coli elongator tRNA(Ala). The other has a sequence similar to Escherichia coli initiator tRNA(Met) (tRNA(iAla/AAA)). Although both tRNAs can be used in poly(U)-directed nonenzymatic initiation at 15 mM Mg2+, only the elongator tRNA can serve for peptide elongation and polyalanine synthesis. Only the initiator tRNA can be bound to 30S ribosomal subunits or 70S ribosomes in the presence of initiation factor 2 (IF-2) and low Mg2+ suggesting that it can function in enzymatic peptide initiation. A derivative of coumarin was covalently attached to the alpha amino group of alanine of these two Ala-tRNA species. The fluorescence spectra, quantum yield and anisotropy for the two Ala-tRNA derivatives are different when they are bound to 70S ribosomes (nonenzymatically in the presence of 15 mM Mg2+) indicating that the local environment of the probe is different. Also, the effect of erythromycin on their fluorescence is quite different, suggesting that the probes and presumably the alanine moiety to which they are covalently linked are in different positions on the ribosomes.

Neighbourhood of the central fold of the tRNA molecule bound to theE.coliribosome—affinity labeling studies with modified tRNAs carrying photoreactive probes attached to the dihydrouridine loop

The neighbourhood of the dihydrouridine loop of tRNA molecule bound to E. coli ribosome has been studied by affinity labeling, using modified tRNAs carrying photoreactive azidonitrophenyl probes attached to the 3-(3-amino-3-carboxypropyl)-uridine located at position 20:1 of Lupin methionine elongator tRNA. The maximum distance between the pyrimidine ring and the azido group estimated for the two probes employed in this study is 10-11 A and 18-19 A, respectively. Cross-linking of the uncharged, modified tRNAs has been studied with poly(A, U, G) as a message, under conditions directing uncharged tRNAs preferentially to the ribosomal P-site. Modified tRNAs bind covalently to both ribosomal subunits with high yields upon irradiation of the respective non-covalent complexes. Proteins S7, L33 and L1 have been consistently found cross-linked to tRNAs modified with both probes, and S5 and L5 to tRNA modified with the longer probe. Surprisingly, an S5-tRNA cross-linking product is reproducibly found in a protein fraction prepared from the purified 50S subunit. Cross-linking to rRNAs is significant only for the longer probe and is stimulated 2-4 fold in the presence of poly(A,U,G). The cross-linking sites are located between nucleotides 1302 and 1398 in 16S rRNA and between nucleotides 2281 and 2358 in 23S rRNA.

Domains of initiator tRNA and initiation codon crucial for initiator tRNA selection by Escherichia coli IF3.

Initiation factors are used by Escherichia coli to select the initiator tRNA over elongator tRNAs during translation initiation. IF3 appears to "inspect" the anticodon end of the tRNA, probably along with the initiation codon. The anticodon stem and loop of the initiator tRNA, together with part of the initiation codon of the mRNA, can be thought of as a unit. Changes made in the anticodon stem, the anticodon loop, or the anticodon of an initiator tRNA fragment result in a loss of selection by IF3 in an in vitro assay for translation initiation. IF3 allows the selection of an initiator tRNA anticodon stem and loop fragment on GUG and UUG codons but does not select that tRNA fragment in response to AUU.