Discrimination between initiation and elongation of protein biosynthesis in yeast: identity assured by a nucleotide modification in the initiator tRNA.

Cytoplasmic initiator tRNAs from plants and fungi possess an unique 2'-phosphoribosyl residue at position 64 of their sequence. In yeast tRNA(iMet), this modified nucleotide located in the T-stem of the tRNA is a 2'-1'-(beta-O-ribofuranosyl-5'-phosphoryl)-adenosine. The phosphoribosyl residue of this modified nucleoside was removed chemically by treatment involving periodate oxidation of tRNA(iMet) and regeneration of the 3'-terminal adenosine with ATP (CTP):tRNA nucleotidyl transferase. The role of phosphoribosylation at position 64 for interaction with elongation factor eEF-1 alpha and initiation factor 2 (eIF-2) was investigated in the homologous yeast system. Whereas the 5'-phosphoribosyl residue prevents the binding of Met-tRNA(iMet) to eEF-1 alpha, it does not influence the interaction with eIF-2. After removal of the ribosyl group, the demodified initiator tRNA showed binding to eEF-1 alpha, but no change was detected with respect to the interaction with the initiation factor eIF-2. This observation is interpreted to mean that a single modification of an eucaryotic initiator tRNA in yeast serves as a negative discriminant for eEF-1 alpha, thus preventing the initiator tRNA(iMet) from entering the elongation cycle of protein biosynthesis.     

Pyrophosphorolysis of CCA addition: implication for fidelity

In nucleic acid polymerization reaction, pyrophosphorolysis is the reversal of nucleotide addition, in which the terminal nucleotide is excised in the presence of inorganic pyrophosphate (PPi). The CCA enzymes are unusual RNA polymerases, which catalyze CCA addition to positions 74-76 at the tRNA 3′ end without using a nucleic acid template. To better understand the reaction mechanism of CCA addition, we tested pyrophosphorolysis of CCA enzymes, which are divided into two structurally distinct classes. Here, we show that only class II CCA enzymes catalyze pyrophosphorolysis and that the reaction can initiate from all three CCA positions and proceed processively until the removal of nucleotide C74. Pyrophosphorolysis of class II enzymes establishes a fundamental difference from class I enzymes, and it is achieved only with the tRNA structure and with specific divalent metal ions. Importantly, pyrophosphorolysis enables class II enzymes to efficiently remove an incorrect A75 nucleotide from the 3′ end, at a rate much faster than the rate of A75 incorporation, suggesting the ability to perform a previously unexpected quality control mechanism for CCA synthesis. Measurement of kinetic parameters of the class II Escherichia coli CCA enzyme reveals that the enzyme catalyzes pyrophosphorolysis slowly relative to the forward nucleotide addition and that it exhibits weak binding affinity to PPi relative to NTP, suggesting a mechanism in which PPi is rapidly released after each nucleotide addition as a driving force to promote the forward synthesis of CCA.     

Functional importance of the 3'-terminal adenosine of tRNA in ribosomal translation

The universally conserved 3'-terminal CCA sequence of tRNA interacts with large ribosomal subunit RNA during translation. The functional importance of the interaction between the 3'-terminal nucleotide of tRNA and the ribosome was studied in vitro using mutant in vitro transcribed tRNA(Val) A76G. Val-tRNA(CCG) does not support polypeptide synthesis on poly(GUA) as a message. However, in a co-translation system, where Val-tRNA(CCG) represented only a small fraction of total Val-tRNA, the mutant tRNA is able to transfer valine into a polypeptide chain, albeit at a reduced level. The A76G mutation does not affect binding of Val- or NAcVal-tRNA(CCG) to the A- or P-sites as shown by efficient peptide bond formation, although the donor activity of the mutant NAcVal-tRNA(CCG) in the peptidyl transfer reaction is slightly reduced compared with wild-type NAcVal-tRNA. Translocation of 3'-CCG-tRNA from the P- to the E-site is not significantly influenced. However, the A76G mutation drastically inhibits translocation of peptidyl-tRNA G(76) from the ribosomal A-site to the P-site, which apparently explains its failure to support cell-free protein synthesis. Our results indicate that the identity of the 3'-terminal nucleotide of tRNA is critical for tRNA movement in the ribosome.     

Formylatable methionine transfer RNA from Mycoplasma: purification and comparison of partial nucleotide sequences with those of other prokaryotic initiator tRNAs.

The major species of the formylatable methionine tRNA from Mycoplasma mycoides var capri has been purified. The 5'- and 3'-terminal sequences of the purified tRNA are pC-G- and C-A-A-C-C-AOH, respectively. Thus, this tRNA also contains the unique structural feature found in two other prokaryotic initiator tRNAs in that the first nucleotide at the 5'-end cannot form a Watson-Crick type of base-pair to the fifth nucleotide from the 3'-end. The Mycoplasma tRNA does not contain ribothymidine; however, a specific uridine residue in the sequence G-U-psi-C-G- can be enzymatically methylated by E. coli extracts to yield G-T-psi-C-G. Since ribothymidine is absent in crude tRNA from this strain of Mycoplasma, the absence of T is probably due to the lack of a U yields T modifying enzyme.     

Identification of in vivo processing intermediates and of splice junctions of tRNAs from maize chloroplasts by amplification with the polymerase chain reaction.

Total RNA from chloroplasts of maize seedlings was used for polymerase chain reaction (PCR) mediated amplification of tRNA precursors and of mature tRNAs encoded by the two split tRNA genes of the ribosomal spacer (tRNA(lle)GAU and tRNA(Ala)UGC) and the single intron-containing tRNA(Gly)UCC gene. Sequence analysis of DNAs amplified from the mature tRNAs by combinations of exon specific primers allows unambiguous identification of the respective splice junctions. Primer combinations in which 5'- or 3'-flanking precursor tRNA sequences are included, leads to the amplification of processing intermediates in which 5'-terminal extensions are still present, whereas no PCR products corresponding to 3'-terminal extensions could be detected. From this it is concluded that in chloroplasts the 5'-terminal endonucleolytic cleavage by RNase P occurs as one of the final steps in the tRNA processing pathway of which the endonucleolytic cleavage at the 3' side probably occurs prior to the splicing of the intron sequences.     

大麦条纹花叶病毒新疆株(BSMV-XJ)RNA2 3′端结构分析

本文利用双脱氧序列分析法对我国大麦条纹花叶病毒新疆株(BSMV-XJ)RNA2 cDNA的3′端进行序列分析,证明XJ株RNA2 3′端239个核苷酸与国外典型株3′端相应部位有高度的序列同源性。通过序列分析及使用寡核苷酸定位裂解法和分子杂交确定,在紧邻239个核苷酸的上游有一个Poly(A)结构,3′终端为一个类tRNA结构,亦与国外典型株相同。经分析认为BSMV-XJ3个基因组RNA具有相同的3′端结构。     

HeLa cells contain a 2'-phosphate-specific phosphotransferase similar to a yeast enzyme implicated in tRNA splicing.

We have previously shown that HeLa cells contain activities implicated in tRNA splicing in yeast, a ligase capable of joining tRNA half-molecules and an NAD-dependent activity capable of removing the 2'-phosphate created at the splice junction by the ligase (Zillmann, M., Gorovsky, M.A., and Phizicky, E.M. (1991) Mol. Cell. Biol. 11, 5410-5416). We show here that removal of the splice junction 2'-phosphate is, as in yeast, a 2'-phosphate-specific phosphotransfer reaction that produces the same, as yet unidentified, small molecule. This enzyme is highly specific for oligomeric substrates having internal 2'-phosphates. Oligomers bearing terminal 2'-phosphates are at least 50-fold less reactive and those bearing 5'- or 3'-terminal phosphates are at least 600-fold less reactive. The requirement for an internal 2'-phosphate can be satisfied by a substrate as small as a dimer.     

The influence of diphosphonic analogues of inorganic pyrophosphate on reactions catalyzed by DNA-dependent RNA-polymerase II

Diphosphonic analogues of inorganic pyrophosphate were studied for their influence both on RNA pyrophosphorolysis and pyrophosphate exchange, catalyzed by purified DNA-dependent RNA-polymerase II from calf thymus. Methylene-, oxyethylene-and aminomethylenediphosphonic acids are shown to compete with PPi for incorporation into nucleoside triphosphate. They activate RNA pyrophosphorolysis in the concentration of 2 mM, but to a less extent than PPi does.     

Sequence-dependent cleavage site selection by RNase Z from the cyanobacterium Synechocystis sp. PCC 6803

Biosynthesis of transfer RNA requires processing from longer precursors at the 5'- and 3'-ends. In eukaryotes, in archaea, and in those bacteria where the 3'-terminal CCA sequence is not encoded, 3' processing is carried out by the endonuclease RNase Z, which cleaves after the discriminator nucleotide to generate a mature 3'-end ready for the addition of the CCA sequence. We have identified and cloned the gene coding for RNase Z in the cyanobacterium Synechocystis sp. PCC 6803. The gene has been expressed in Escherichia coli, and the recombinant protein was purified. The enzymatic activity of RNase Z from Synechocystis has been studied in vitro with a variety of substrates. The presence of C or CC after the discriminator nucleotide modifies the cleavage site of RNase Z so that it is displaced by one and two nucleotides to the 3'-side, respectively. The presence of the complete 3'-terminal CCA sequence in the precursor of the tRNA completely inhibits RNase Z activity. The inactive CCA-containing precursor binds to Synechocystis RNase Z with similar affinity than the mature tRNA. The properties of the enzyme described here could be related with the mechanism by which CCA is added in this organism, with the participation of two separate nucleotidyl transferases, one specific for the addition of C and another for the addition of A. This work is the first characterization of RNase Z from a cyanobacterium, and the first from an organism with two separate nucleotidyl transferases.     

Preparation of 2-azidoadenosine 3',5'-[5'-32P]bisphosphate for incorporation into transfer RNA. Photoaffinity labeling of Escherichia coli ribosomes

2-Azidoadenosine was synthesized from 2-chloroadenosine by sequential reaction with hydrazine and nitrous acid and then bisphosphorylated with pyrophosphoryl chloride to form 2-azidoadenosine 3',5'-bisphosphate. The bisphosphate was labeled in the 5'-position using the exchange reaction catalyzed by T4 polynucleotide kinase in the presence of [gamma-32P]ATP. Polynucleotide kinase from a T4 mutant which lacks 3'-phosphatase activity (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) was required to facilitate this reaction. 2-Azidoadenosine 3',5'-[5'-32P]bisphosphate can serve as an efficient donor in the T4 RNA ligase reaction and can replace the 3'-terminal adenosine of yeast tRNAPhe with little effect on the amino acid acceptor activity of the tRNA. In addition, we show that the modified tRNAPhe derivative can be photochemically cross-linked to the Escherichia coli ribosome.     

Crosslinking of mRNA analog, pUUAGUAUUUAUU derivative with a photoactivated group at the guanosine residue, with human 80S ribosomes

Crosslinking of mRNA analog, dodecaribonucleotide pUUAGUAUUUAUU derivative carrying a perfluoroarylazido group at the guanine N7, was studied in model complexes with 80S ribosomes involving tRNA and in binary complex (i.e., in the absence of tRNA). It was shown that, irrespectively of complex formation conditions (13 mM Mg2+, or 4 mM Mg2+ in the presence of polyamines), the mRNA analog in binary complex with 80S ribosomes was crosslinked with sequence 1840-1849 of 18S rRNA, but in the complexes formed with participation of Phe-TPHKPhe (where the G residue carrying the arylazido group occupied position-3 to the first nucleotide of the UUU codon at the P site) the analog was crosslinked with nucleotide 1207. The presence and the nature of tRNA at the E site had no effect on the environment of position-3 of the mRNA analog. Efficient crosslinking of the mRNA analog with tRNA was observed in all studied types of complex. Modified codon GUA, when located at the E site, underwent crosslinking with both cognate valine tRNA and noncognate aspartate tRNA for which the extent of binding at the E site of 80S ribosomes was almost the same and depended little on Mg2+ concentration and the presence of polyamines.     

E. coli initiator tRNA analogs with different nucleotides in the discriminator base position.

The effect of base changes at the fourth position from the 3'-terminus of Escherichia coli initiator tRNAMet has been studied to test the 'discriminator hypothesis' which proposed that the nucleotide in this position might have a role in the specificity of the aminoacylation reaction. E. coli initiator tRNA lacking the 3'-terminal tetranucleotide was prepared by partial digestion with S1 nuclease. To construct tRNA analogs with different bases in the fourth position this truncated tRNA was joined by RNA ligase to each of four chemically synthesized 2',3'-ethoxy-methylidene tetranucleotides pACCA(em), pCCCA(em), pGCCA(em), and pUCCA(em). In vitro aminoacylation studies showed that all four molecules accepted methionine, albeit with different Vmax values.     

Preparation of Escherichia coli tRNAs terminating of modified nucleosides by the use of CTP(ATP):tRNA nucleotidyltransferase and polynucleotide phosphorylase.

Two procedures were investigated for the modification of tRNAs at the 3'-terminal nucleoside. The first involved the incubation of an enzymatically abreviated tRNA (tRNA-C-COH) with appropriate nucleoside triphosphates in the presence of CTP(ATP):tRNA nucleotidyltransferase from Escherichia coli and yeast. The E. coli enzyme did not utilize 2'- or 3'-deoxyadenosine 5'-triphosphate as substrates, but affected incorporation of the 2'- and 3'-O-methyladenosine triphosphates onto tRNA-C-Cou to the extent of 30 and 37%, respectively. Although incorporation of the deoxynucleotides could not be effected using the E. coli enzyme, yeast CTP(ATP:tRNA nucleotidyltransferase produced the desired tRNAs in yields of 45-65%. The second modification procedure involved incubation of tRNA-C-COH with (appropriately blocked) nucleoside diphosphates in the presence of polynucleotide phosphorylase. This procedure afforded the tRNAs terminating in 2'- and 3'-deoxyadenosine in yields of 4% (and the yield of the former was increased to 36% when the incubation was carried out in the presence of 20% methanol). The yields of tRNAs terminating in 2'- and 3'-O-methyladenosing produced by this procedure were 55 and 17%, respectively. Because only single isomers of most of the tRNAs terminating in 2'- and 3'-deoxy- and O-methyladenosine are aminoacylated, attempts were made to obtain the other isomericaminoacyl-tRNA by enzymatic introduction of chemically preaminoacylated nucleotides onto tRNA-C-COH. Although incubation of tRNA-C-COH with three aminoacylated nucleoside 5'-triphosphates and E. coli CTP(ATP):tRNA nucleotidyltransferase did not result in production of the desired tRNAs to a detectable extent, incubation with 2'-deoxy-3'-O-L-phenylalanyladenosine 5'-diphosphate and polynucleotide phosphorylase afforded E. coli tRNA terminating with the corresponding aminoacylated deoxynucleoside.     

Responsibility of tRNA(Ile) for spermine stimulation of rat liver Ile-tRNA formation

To determine whether tRNA or aminoacyl-tRNA synthetase is responsible for spermine stimulation of rat liver Ile-tRNA formation, homologous and heterologous Ile-tRNA formations were carried out with Escherichia coli and rat liver tRNA(Ile) and their respective purified Ile-tRNA synthetases. Spermine stimulation was observed only when tRNA from the rat liver was used. Spermine bound to rat liver tRNA(Ile) but not to the purified aminoacyl-tRNA synthetase complex. Kinetic analysis of Ile-tRNA formation revealed that spermine increased the Vmax and Km values for rat liver tRNA(Ile). The Km value for ATP and isoleucine did not change significantly in the presence of spermine. Furthermore, higher concentrations of rat liver tRNA(Ile) tended to inhibit Ile-tRNA formation if spermine was absent. Spermine restored isoleucine-dependent PPi-ATP exchange in the presence of rat liver tRNA(Ile), an inhibitor of this exchange. The nucleotide sequence of rat liver tRNA(Ile) was determined and compared with that of E. coli tRNA(Ile). Differences in nucleotide sequences of the two tRNAs(Ile) were observed mainly in the acceptor and anticodon stems. Limited ribonuclease V1 digestion of the 3'-32P-labeled rat liver tRNA(Ile) showed that both the anticodon and acceptor stems were structurally changed by spermine, and that the structural change by spermine was different from that by Mg2+. The influence of spermine on the ribonuclease V1 digestion of E. coli tRNA(Ile) was different from that of rat liver tRNA(Ile). The results suggest that the interaction of spermine with the acceptor and anticodon stems may be important for spermine stimulation of rat liver Ile-tRNA formation.     

Specific recognition of the 3'-terminal adenosine of tRNAPhe in the exit site of Escherichia coli ribosomes

Ribosomes from Escherichia coli possess, in addition to A and P sites, a third tRNA binding site, which according to its presumed function in tRNA release during translocation has been termed the exit site. The exit site exhibits a remarkable specificity for deacylated tRNA; charged tRNA, e.g. N-AcPhe-tRNAPhe, is not bound significantly. To determine the molecular basis of this discrimination, we have measured the exit site binding affinities of a number of derivatives of tRNAPhe from E. coli, modified at the 3' end. Binding to the exit site of the tRNAPhe derivatives was measured fluorimetrically by competition with a fluorescent tRNAPhe derivative. We show here that removal of the 2' and 3' hydroxyl groups of the 3'-terminal adenosine decreases the affinity of tRNAPhe for the exit site 15 and 40-fold, respectively. Substitutions at the 3' hydroxyl group (aminoacylation, phosphorylation, cytidylation) as well as removal of the 3'-terminal adenosine (or adenylate) of tRNAPhe lower the affinity below the detection limit of 2 x 10(5) M-1, i.e. more than 100-fold. Modification of the adenine moiety (1,N6-etheno adenine) or replacement of it with other bases (cytosine, guanine) has the same dramatic effect. In contrast, the binding to both P and A sites is virtually unaffected by all of the modifications tested. These results suggest that a major fraction (at least -12 kJ/mol, probably about -17 kJ/mol) of the free energy of exit site binding of tRNAPhe (-42 kJ/mol at 20 mM-Mg2+) is contributed by the binding of the 3'-terminal adenine to the ribosome. The binding most likely entails the formation of hydrogen bonds.