The use of cross-linking platinum reagents in the study of tRNA and mRNA interaction with ribosomes

The use of some bifunctional Pt(II)-containing cross-linking reagents for investigation of structural organization of ribosomal tRNA- and mRNA-binding centres is demonstrated for various types of [70S ribosome.mRNA-tRNA] complexes. It is shown that treatment of the complexes [70S ribosome.Ac[14C]Phe-tRNA(Phe).poly(U)], [70S ribosome.3'-32pCp-tRNA(Phe).poly(U)] and [70S ribosome.f[35S]Met-tRNA(fMet).AUGU6] with Pt(II)-derivatives results in covalent attachment of tRNA to ribosome. AcPhe-tRNA(Phe) and 3'-pCp-tRNA(Phe) bound at the P site were found to be cross-linked preferentially to 30S subunit. fMet-tRNA(fMet) within the 70S initiation complex is cross-linked to both ribosome subunits approximately in the same extent, which exceeds two-fold the level of the tRNA(Phe) cross-linking. All used tRNA species were cross-linked in the comparable degree both to rRNA and proteins of both subunits in all types of the complexes studied. 32pAUGU6 cross-links exclusively to 30S subunit (to 16S RNA only) within [70S ribosome.32pAUGU6.fMet-tRNA(fMet)] complex. In the absence of fMet-tRNAfMet the level of the cross-linking is 4-fold lower.     

Initiation of protein synthesis in Saccharomyces cerevisiae mitochondria without formylation of the initiator tRNA

Protein synthesis in eukaryotic organelles such as mitochondria and chloroplasts is widely believed to require a formylated initiator methionyl tRNA (fMet-tRNA(fMet)) for initiation. Here we show that initiation of protein synthesis in yeast mitochondria can occur without formylation of the initiator methionyl-tRNA (Met-tRNA(fMet)). The formylation reaction is catalyzed by methionyl-tRNA formyltransferase (MTF) located in mitochondria and uses N(10)-formyltetrahydrofolate (10-formyl-THF) as the formyl donor. We have studied yeast mutants carrying chromosomal disruptions of the genes encoding the mitochondrial C(1)-tetrahydrofolate (C(1)-THF) synthase (MIS1), necessary for synthesis of 10-formyl-THF, and the methionyl-tRNA formyltransferase (open reading frame YBL013W; designated FMT1). A direct analysis of mitochondrial tRNAs using gel electrophoresis systems that can separate fMet-tRNA(fMet), Met-tRNA(fMet), and tRNA(fMet) shows that there is no formylation in vivo of the mitochondrial initiator Met-tRNA in these strains. In contrast, the initiator Met-tRNA is formylated in the respective "wild-type" parental strains. In spite of the absence of fMet-tRNA(fMet), the mutant strains exhibited normal mitochondrial protein synthesis and function, as evidenced by normal growth on nonfermentable carbon sources in rich media and normal frequencies of generation of petite colonies. The only growth phenotype observed was a longer lag time during growth on nonfermentable carbon sources in minimal media for the mis1 deletion strain but not for the fmt1 deletion strain.     

Purification and characterization of yeast mitochondrial initiation factor 2

Yeast mitochondrial initiation factor 2 (ymIF2) is encoded by the nuclear IFM1 gene. A His-tagged version of ymIF2, lacking its predicted mitochondrial presequence, was expressed in Escherichia coli and purified. Purified ymIF2 bound both E. coli fMet-tRNA(f)(Met) and Met-tRNA(f)(Met), but binding of formylated initiator tRNA was about four times higher than that of the unformylated species under the same conditions. In addition, the isolated ymIF2 was compared to E. coli IF2 in four other assays commonly used to characterize this initiation factor. Formylated and nonformylated Met-tRNA(f)(Met) were bound to E. coli 30S ribosomal subunits in the presence of ymIF2, GTP, and a short synthetic mRNA. The GTPase activity of ymIF2 was found to be dependent on the presence of E. coli ribosomes. The ymIF2 protected fMet-tRNA(f)(Met) to about the same extent as E. coli IF2 against nonenzymatic deaminoacylation. In contrast to E. coli IF2, the complex formed between ymIF2 and fMet-tRNA(f)(Met) was not stable enough to be analyzed in a gel shift assay. In similarity to other IF2 species isolated from bacteria or bovine mitochondria, the N-terminal domain could be eliminated without loss of initiator tRNA binding activity.     

Purification and some properties of a protein binding and deacylating initiator transfer ribonucleic acid

1. A protein factor promoting the binding of initiator tRNA to the 40S ribosomal subunit was purified to homogeneity (more than 2500-fold) from rat liver cytosol. It has a mol.wt. of 265000 and is composed of four subunits of identical molecular weight. 2. This factor directs the binding of methionyl-tRNA(fMet) and to a lesser extent also of N-acetylphenylalanyl-tRNA, but not of methionyl-tRNA(Met) or phenylalanyl-tRNA, to the smaller ribosomal subunit at high concentrations of GTP (8-10mm) with an optimum at pH4.0. As evidenced by sucrose-density-gradient centrifugation, initiator tRNA becomes bound to the 40S subunit or to 80S ribosomes. 3. A deacylase activity specific for methionyl-tRNA(fMet) is associated with the pure factor. The factor significantly stimulates the translation of natural message in systems containing polyribosomes and both purified peptide-elongation factors. 4. The factor binds initiator tRNA or GTP to form unstable binary complexes and forms a ternary complex with methionyl-tRNA(fMet) and GTP. This complex is relatively stable. 5. In the absence of any cofactors the factor forms a stable complex with 40S and 80S ribosomes. This preformed ribosomal complex binds efficiently initiator tRNA at pH7.5 and low concentrations of GTP (1-2mm). The ternary complex of the factor with methionyl-tRNA(fMet) and GTP may be liberated from this ribosomal complex. 6. A protein factor capable of promoting the binding and simultaneously the deacylation of initiator tRNA may apparently have a regulatory function in physiological gene translation by removing an excess of methionyl-tRNA(fMet) not required for translation.     

A quantitative kinetic scheme for 70 S translation initiation complex formation

Association of the 30 S initiation complex (30SIC) and the 50 S ribosomal subunit, leading to formation of the 70 S initiation complex (70SIC), is a critical step of the translation initiation pathway. The 70SIC contains initiator tRNA, fMet-tRNA(fMet), bound in the P (peptidyl)-site in response to the AUG start codon. We have formulated a quantitative kinetic scheme for the formation of an active 70SIC from 30SIC and 50 S subunits on the basis of parallel rapid kinetics measurements of GTP hydrolysis, Pi release, light-scattering, and changes in fluorescence intensities of fluorophore-labeled IF2 and fMet-tRNA(f)(Met). According to this scheme, an initially formed labile 70 S complex, which promotes rapid IF2-dependent GTP hydrolysis, either dissociates reversibly into 30 S and 50 S subunits or is converted to a more stable form, leading to 70SIC formation. The latter process takes place with intervening conformational changes of ribosome-bound IF2 and fMet-tRNA(fMet), which are monitored by spectral changes of fluorescent derivatives of IF2 and fMet-tRNA(fMet). The availability of such a scheme provides a useful framework for precisely elucidating the mechanisms by which substituting the non-hydrolyzable analog GDPCP for GTP or adding thiostrepton inhibit formation of a productive 70SIC. GDPCP does not affect stable 70 S formation, but perturbs fMet-tRNA(fMet) positioning in the P-site. In contrast, thiostrepton severely retards stable 70 S formation, but allows normal binding of fMet-tRNA(fMet)(prf20) to the P-site.     

Evidence for a tRNA/rRNA interaction site within the peptidyltransferase center of the Escherichia coli ribosome

A nine-base oligodeoxyribonucleotide complementary to bases 2497-2505 of 23S rRNA was hybridized to both 50S subunits and 70S ribosomes. The binding of the probe to the ribosome or ribosomal subunits was assayed by nitrocellulose filtration and by sucrose gradient centrifugation techniques. The location of the hybridization site was determined by digestion of the rRNA/cDNA heteroduplex with ribonuclease H and gel electrophoresis of the digestion products, followed by the isolation and sequencing of the smaller digestion fragment. The cDNA probe was found to interact specifically with its rRNA target site. The effects on probe hybridization to both 50S and 70S ribosomes as a result of binding deacylated tRNA(Phe) were investigated. The binding of deacylated tRNA(Phe), either with or without the addition of poly(uridylic acid), caused attenuation of probe binding to both 50S and 70S ribosomes. Probe hybridization to 23S rRNA was decreased by about 75% in both 50S subunits and 70S ribosomes. These results suggest that bases within the 2497-2505 site may participate in a deacylated tRNA/rRNA interaction.     

Inhibition of bacterial IF2 binding to fMet-tRNA((fMet)) by aminoglycosides

Screening for inhibitors of bacterial protein synthesis Initiation Factor 2 (IF2) binding to N-formyl-Methionyl-transfer RNA (fMet-tRNA((fMet))) identified a series of aminoglycosides, that included amikacin and kanamycin A1, as inhibitors of this interaction. Subsequent testing revealed that aminoglycosides displayed a wide range of inhibitory activity. However, the failure of these compounds to completely inhibit binding of IF2 to fMet-tRNA((fMet)), the known ability of aminoglycosides to bind RNA, and the ability of the aminoglycosides to displace PicoGreen bound to fMet-tRNA((fMet)) suggest these compounds act by binding fMet-tRNA((fMet)). This hypothesis is further supported by isothermal denaturation experiments that failed to show any interaction between the IF2 protein and the aminoglycosides.     

The conversion of eIF-2.GDP to eIF-2.GTP by eIF-2B requires Met-tRNA(fMet).

We have investigated why the recycling of eIF-2.GDP to eIF-2.GTP, mediated by the guanine nucleotide exchange factor eIF-2B, is rapid in rabbit reticulocyte lysate, reconstituted for optimal protein synthesis, but slow in an isolated reaction with purified eIF-2B. We have found that purified eIF-2B dissociates eIF-2.[3H]GDP as efficiently in the presence of GTP as it does in the presence of GDP provided Met-tRNA(fMet) is added. tRNA(fMet) is ineffective, and there is no Met-tRNA(fMet) requirement for exchange with GDP. Exchange of eIF-2 bound GDP for GTP is completely dependent upon Met-tRNA(fMet) in the presence of ATP, suggesting that under physiological conditions efficient recycling of eIF-2.GDP to eIF-2.GTP requires conversion of the latter, a relatively unstable complex, to a more stable Met-tRNA(fMet).eIF-2.GTP complex.     

Codon-specific interaction of uncharged transfer-RNA with eukaryotic ribosomes.

Rat liver ribosomes bound [32P]tRNAPhe in both a codon-dependent and codon-independent manner. The codon-dependent binding was studied further by utilising the ability of the unchanged tRNAPhe to inhibit the poly(U)-directed binding of [3H]Phe-tRNA to ribosomes. At least part of the codon-dependent binding of uncharged tRNA appears to be to the ribosomal A-site.     

Footprinting mRNA-ribosome complexes with chemical probes.

We footprinted the interaction of model mRNAs with 30S ribosomal subunits in the presence or absence of tRNA(fMet) or tRNA(Phe) using chemical probes directed at the sugar-phosphate backbone or bases of the mRNAs. When bound to the 30S subunits in the presence of tRNA(fMet), the sugar-phosphate backbones of gene 32 mRNA and 022 mRNA are protected from hydroxyl radical attack within a region of about 54 nucleotides bounded by positions -35 (+/- 2) and +19, extending to position +22 when tRNA(Phe) is used. In 70S ribosomes, protection is extended in the 5' direction to about position -39 (+/- 2). In the absence of tRNA, the 30S subunit protects only nucleotides -35 (+/- 2) to +5. Introduction of a stable tetraloop hairpin between positions +10 and +11 of gene 32 mRNA does not interfere with tRNA(fMet)-dependent binding of the mRNA to 30S subunits, but results in loss of protection of the sugar-phosphate backbone of the mRNA downstream of position +5. Using base-specific probes, we find that the Shine-Dalgarno sequence (A-12, A-11, G-10 and G-9) and the initiation codon (A+1, U+2 and G+3) of gene 32 mRNA are strongly protected by 30S subunits in the presence of initiator tRNA. In the presence of tRNA(Phe), the same Shine-Dalgarno bases are protected, as are U+4, U+5 and U+6 of the phenylalanine codon. Interestingly, A-1, immediately preceding the initiation codon, is protected in the complex with 30S subunits and initiator tRNA, while U+2 and G+3 are protected in the complex with tRNA(Phe) in the absence of initiator tRNA. Additionally, specific bases upstream from the Shine-Dalgarno region (U-33, G-32 and U-22) as well as 3' to the initiation codon (G+11) are protected by 30S subunits in the presence of either tRNA. These results imply that the mRNA binding site of the 30S subunit covers about 54-57 nucleotides and are consistent with the possibility that the ribosome interacts with mRNA along its sugar-phosphate backbone.     

Properties of elongation factor G: its interaction with the ribosomal peptidyl-site

EF-G bound to poly(U)·ribosomes prevents enzymatic or nonenzymatic binding of charged tRNA not only to the A-site but also to the P-site. In turn, charged tRNA bound either to the P- or A-site prevents formation of EF-G·GMPPCP·ribosome complex. Ribosomes carrying newly synthetized peptidyl-tRNA in pretranslocative state are also unable to form stable complexes with EF-G. The functional implications of these observations are discussed and it is suggested that tRNA plays a regulatory role in the interaction of EF-G with ribosomes during the cyclic process of elongation.     

Mapping the fMet-tRNA(f)(Met) binding site of initiation factor IF2

The interaction between fMet-tRNA(f)(Met) and Bacillus stearothermophilus translation initiation factor IF2 has been characterized. We demonstrate that essentially all thermodynamic determinants governing the stability and the specificity of this interaction are localized within the acceptor hexanucleotide fMet-3'ACCAAC of the initiator tRNA and a fairly small area at the surface of the beta-barrel structure of the 90-amino acid C-terminal domain of IF2 (IF2 C-2). A weak but specific interaction between IF2 C-2 and formyl-methionyl was also demonstrated. The surface of IF2 C-2 interacting with fMet-tRNA(f)(Met) has been mapped using two independent approaches, site- directed mutagenesis and NMR spectroscopy, which yielded consistent results. The binding site comprises C668 and G715 located in a groove accommodating the methionyl side-chain, R700, in the vicinity of the formyl group, Y701 and K702 close to the acyl bond between fMet and tRNA(f)(Met), and the surface lined with residues K702-S660, along which the acceptor arm of the initiator tRNA spans in the direction 3' to 5'.     

Relationship between size of mRNA ribosomal binding site and initiation factor function

The rate and the extent of the binding of initiator fMet-tRNA(fMet) to 30S ribosomal subunits in the presence of IF1, IF2 and GTP is either inhibited or slightly stimulated by the presence of IF3 depending on whether the initiation triplet AUG or the polynucleotide poly(AUG) is used as template. To determine the length of the template required for the transition from the AUG- to the poly(AUG)-type of behavior in the presence of IF3, the ribosomal binding of fMet-tRNA was studied in response to AUG triplets extended on either the 5'- or the 3'-side by stretches of homo-oligonucleotides of different lengths. When the binding of fMet-tRNA was studied at equilibrium it was found that IF3 no longer inhibits the amount of ternary complex formed if AUG is extended either 10 nucleotides on the 5'- or 35-40 nucleotides on the 3'-side. When the initial rate of ternary complex formation is considered, shorter extensions (4 nucleotides on the 5'-side or 20-30 nucleotides on the 3'-side) are sufficient to elicit a substantial stimulation by IF3. These results are discussed in relation to the mechanism of action of the initiation factors in the selection of the initiation region of the mRNA by ribosomes.     

Interaction of ribo- and deoxyriboanalogs of yeast tRNA(Phe) anticodon arm with programmed small ribosomal subunits of Escherichia coli and rabbit liver.

A synthetic ribooligonucleotide, r(CCAGACUGm-AAGAUCUGG), corresponding to the unmodified yeast tRNA(Phe) anticodon arm is shown to bind to poly(U) programmed small ribosomal subunits of both E. coli and rabbit liver with affinity two order less than that of a natural anticodon arm. Its deoxyriboanalogs d(CCAGACTGAAGATCTGG) and d(CCAGA)r(CUGm-AAGA)d(TCTGG), are used to study the influence of sugar-phosphate modification on the interaction of tRNA with programmed small ribosomal subunits. The deoxyribooligonucleotide is shown to adopt a hairpin structure. Nevertheless, as well as oligonucleotide with deoxyriboses in stem region, it is not able to bind to 30S or 40S ribosomal subunits in the presence of ribo-(poly(U] or deoxyribo-(poly (dT) template. The deoxyribooligonucleotide also has no inhibitory effect on tRNA(Phe) binding to 30S ribosomes at 10-fold excess over tRNA. Neomycin does not influence binding of tRNA anticodon arm analogs used. Complete tRNA molecule and natural modifications of anticodon arm are considered to stabilize the arm structure needed for its interaction with a programmed ribosome.     

Further evidence for the participation of proteins S 3, S 14 and S 19 in tRNA binding to E. coli 30 S subunits

Previous studies have shown that iodination of 30 S subunits causes inactivation for both enzymatic fMet-tRNA and non-enzymatic phe-tRNA binding activities. This inactivation was shown to be due to the modification of three to five ribosomal proteins [1]. In this report the role of these proteins in tRNA binding activity has been further studied. Purified ribosomal proteins, isolated from modified subunits, are re-assembled into otherwise unmodified 30 S ribosomes and assayed for tRNA binding capacity. The presence of modified S 3, S 14 and S 19 (S 15) in the reconstituted particle results in substantial reduction of both fMet-tRNA and phe-tRNA binding activities. This reduction in tRNA binding activity does not appear to be due to an assembly defect.     

Characterization of 16S rRNA mutations that decrease the fidelity of translation initiation

In bacteria, initiation of translation is kinetically controlled by factors IF1, IF2, and IF3, which work in conjunction with the 30S subunit to ensure accurate selection of the initiator tRNA (fMet-tRNA(fMet)) and the start codon. Here, we show that mutations G1338A and A790G of 16S rRNA decrease initiation fidelity in vivo and do so in distinct ways. Mutation G1338A increases the affinity of tRNA(fMet) for the 30S subunit, suggesting that G1338 normally forms a suboptimal Type II interaction with fMet-tRNA(fMet). By stabilizing fMet-tRNA(fMet) in the preinitiation complex, G1338A may partially compensate for mismatches in the codon-anti-codon helix and thereby increase spurious initiation. Unlike G1338A, A790G decreases the affinity of IF3 for the 30S subunit. This may indirectly stabilize fMet-tRNA(fMet) in the preinitiation complex and/or promote premature docking of the 50S subunit, resulting in increased levels of spurious initiation.     

Binding of ribosome recycling factor to ribosomes,comparison with tRNA

The prokaryotic post-termination ribosomal complex is disassembled by ribosome recycling factor (RRF) and elongation factor G. Because of the structural similarity of RRF and tRNA, we compared the biochemical characteristics of RRF binding to ribosomes with that of tRNA. Unesterified tRNA inhibited the disassembly of the post-termination complex in a competitive manner with RRF, suggesting that RRF binds to the A-site. Approximately one molecule of ribosome-bound RRF was detected after isolation of the RRF-ribosome complex. RRF and unesterified tRNA similarly inhibited the binding of N-acetylphenylalanyl-tRNA to the P-site of non-programmed but not programmed ribosomes. Under the conditions in which unesterified tRNA binds to both the P- and E-sites of non-programmed ribosomes, RRF inhibited 50% of the tRNA binding, suggesting that RRF does not bind to the E-site. The results are consistent with the notion that a single RRF binds to the A- and P-sites in a somewhat analogous manner to the A/P-site bound peptidyl tRNA. The binding of RRF and tRNA to ribosomes was influenced by Mg(2+) and NH(4)(+) ions in a similar manner.     

The Cryo-EM structure of a complete 30S translation initiation complex from Escherichia coli

Formation of the 30S initiation complex (30S IC) is an important checkpoint in regulation of gene expression. The selection of mRNA, correct start codon, and the initiator fMet-tRNA(fMet) requires the presence of three initiation factors (IF1, IF2, IF3) of which IF3 and IF1 control the fidelity of the process, while IF2 recruits fMet-tRNA(fMet). Here we present a cryo-EM reconstruction of the complete 30S IC, containing mRNA, fMet-tRNA(fMet), IF1, IF2, and IF3. In the 30S IC, IF2 contacts IF1, the 30S subunit shoulder, and the CCA end of fMet-tRNA(fMet), which occupies a novel P/I position (P/I1). The N-terminal domain of IF3 contacts the tRNA, whereas the C-terminal domain is bound to the platform of the 30S subunit. Binding of initiation factors and fMet-tRNA(fMet) induces a rotation of the head relative to the body of the 30S subunit, which is likely to prevail through 50S subunit joining until GTP hydrolysis and dissociation of IF2 take place. The structure provides insights into the mechanism of mRNA selection during translation initiation.     

Rapid peptide bond formation on isolated 50S ribosomal subunits

The catalytic site of the ribosome, the peptidyl transferase centre, is located on the large (50S in bacteria) ribosomal subunit. On the basis of results obtained with small substrate analogues, isolated 50S subunits seem to be less active in peptide bond formation than 70S ribosomes by several orders of magnitude, suggesting that the reaction mechanisms on 50S subunits and 70S ribosomes may be different. Here we show that with full-size fMet-tRNA(fMet) and puromycin or C-puromycin as peptide donor and acceptor substrates, respectively, the reaction proceeds as rapidly on 50S subunits as on 70S ribosomes, indicating that the intrinsic activity of 50S subunits is not different from that of 70S ribosomes. The faster reaction on 50S subunits with fMet-tRNA(fMet), compared with oligonucleotide substrate analogues, suggests that full-size transfer RNA in the P site is important for maintaining the active conformation of the peptidyl transferase centre.