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PubMed1976

Changes in tertiary structure accompanying a single base change in transfer RNA. Proton magnetic resonance and aminoacylation studies of Escherichia coli tRNAMet f1 and tRNAMet f3 and their spin-labeled (s4U8) derivatives.

The properties of Escherichia coli tRNAMet f1 and tRNAMet f3 that differ by only one base change, m7G to A at position 47, have been compared structurally by proton magnetic resonance and functionally by the aminoacylation reaction. The NMR spectra of the two tRNA species in the region between 0 and 4 ppm below 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) (methyl and methylene region) were the same except for the absence of the lowest field peak at 3.8 ppm in tRNAMet f3, thus unequivocally identifying this resonance at the methyl group of m7G47 of tRNAMet f1. The same resonance disappears in tRNAMet f1 spin-labeled at s4U8 and reappears in the diamagnetic reduced spin-labeled tRNAMet f1 from which the average distance between the spin-label and the methyl protons of m7G is estimated to be less than 15 A. The proximity of m7G47 but not T55 to s4U8 in the structure of E. coli tRNAMet f1 in solution is consistant with the crystallographic model for yeast tRNAPhe. A spectral comparison of the hydrogen-bond regions (11-14 ppm below DSS) of tRNAMet f1 and tRNAMet f3 reveals major shifts of four resonances previously assigned to tertiary hydrogen bonds. Of the four, the one at lowest field (14.8 ppm) had been assigned by chemical modification to the tertiary (s4U8-A14) hydrogen bond and the one at 13.3 ppm had been tentatively assigned to the tertiary hydrogen bond G23-m7G47 of the 13-23-47 triple. A more positive assignment of the G23-m7G47 at 13.3 ppm could be made from the additional evidence that this resonance, which was first observed in the difference spectrum between spin-labeled tRNAMet f1 and its reduced form, is the only one missing in the analogous difference spectrum of tRNAMet f3. At low ionic strength and in the absence of magnesium ions, the differences in the hydrogen-bonded region of the NMR spectra of tRNAMet f1 and tRNAMet f3 are much greater than in the presence of magnesium ions. The optimal magnesium concentration required for maximal initial velocities is also higher for tRNAMet f3 than for tRNAMet f1. The perturbation caused by the spin-label in destabilizing hydrogen bonds in the region between 13 and 14 ppm is greater for tRNAMet f3 than tRNAMet f1 but the distance relations for the hydrogen bonds in the region between 12 and 13 ppm (the major paramagnetic perturbations) are conserved in the two species. The disruption of one hydrogen bond relative to native tRNAMet f1 either by spin-labeling (s4U8-A14) or by substitution of m7G by A in tRNAMet f3 has little effect on the aminoacyl acceptor activity or the velocity of the aminoacylation reaction at optimal magnesium concentration, but the absence of both tertiary hydrogen bonds in the augmented D-helix region in the spin-labeled tRNAMet f3 results in approximately 60% reduction both in acceptance activity and in initial velocity of the aminoacylation reaction.

Base Sequence↗
PubMed1975

Modification of the 5'-terminus of mRNA by soluble guanylyl and methyl transferases from vaccinia virus.

RNA guanylyl and methyl transferases have been solubilized from vaccinia virus cores. The guanylyl transferase specifically adds a GMP residue to the 5'-terminus of unmethylated vaccinia virus mRNA to form the structures G(5')ppp(5')Gp- and G(5')ppp(5')Ap-. Studies with [alpha-32P]GTP and [beta, gamma-32P]GTP indicated that only the alpha-phosphate is transferred. In the presence of S-adenosylmethionine, the methyl transferases convert the blocked 5'-termini to m7G(5')ppp(5')Gmp- and m7G(5')ppp(5')Amp-. Similarly, the enzymes can modify synthetic poly(A) to form the structure m7G(5')ppp(5')Amp-.

Calcium↗
PubMed1992

Mass spectrometry of mRNA cap 4 from trypanosomatids reveals two novel nucleosides.

Synthesis of mRNA in kinetoplastid protozoa involves the process of trans-splicing, in which an identical 39-41-nucleotide (depending on the species) mini-exon is placed at the 5' end of mature mRNAs. The mini-exon sequence is highly conserved among all members of the Kinetoplastida, nucleotides 1-6 being identical in the four genera so far examined. Prior to trans-splicing, the mini-exon donor RNA is capped by the addition of a (5'-5') triphosphate-linked 7-methylguanosine, followed by modification of the first four transcribed nucleotides. Partial structures have been previously deduced for this cap 4 moiety from Trypanosoma brucei and Leptomonas collosoma. We have purified enough cap 4 from T. brucei and Crithidia fasciculata to allow definitive structural analysis by combined liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry. The results, together with the known mini-exon sequence, show that cap 4 in both species has the structure m7G(5')ppp(5')m6(2)AmpAmpCmpm3Ump. The presence of N6,N6,2'-O-trimethyladenosine and 3,2'-O-dimethyluridine, nucleosides previously unknown in nature, were confirmed by rigorous comparison with synthetic standards. The conservation of cap 4 between these divergent genera suggests that this structure may be common to most if not all Kinetoplastida.

Animals↗
PubMed1983

Enzymatic methylation of chemically alkylated DNA and poly(dG-dC) X poly(dG-dC) in B and Z forms.

The enzymatic methylation of chemically alkylated DNA and of poly(dG-dC) X poly(dG-dC) by beef brain DNA(cytosine-5-)-methyltransferase have been tested. The alkylation by dimethylsulfate, which yields mostly 7 methylguanine (m7G) and 3 methyladenine (m3A) do not affect the enzymatic methylation. The dimethylsulfate alkylated poly(dG-dC) X poly(dG-dC) converted into the Z-form in the presence of MgCl2, is just as well methylated as the native or the alkylated polynucleotide in the B-form. The alkylation of DNA or of poly(dG-dC) X poly(dG-dC) by methylnitrosourea yields, in addition to the above base modifications described for dimethylsulfate, methylphosphotriesters and O6-methylguanine. The enzymatic methylation of these substrates modified by methylnitrosourea is decreased. This decrease is proportional to the extent of the chemical alkylation of the substrate.

Alkylation↗
PubMed1978

HeLa cell RNA (2'-O-methyladenosine-N6-)-methyltransferase specific for the capped 5'-end of messenger RNA.

A novel enzyme involved in the post-transcriptional modification of the 5'-end of mRNA has been partially purified from HeLa cells. Termed an S-adenosyl-L-methionine:RNA(2'-O-methyladenosine-N4)-methyltransferase, the enzyme specifically catalyzes the transfer of a methyl group from S-adenosylmethionine to the N6 position of a 2'-O-methyladenosine residue located within the "capped" 5'-end of mRNA. The dimethylated nucleoside, N6,2'-O-dimethyladenosine, is formed as indicated by the following reaction in which m7G(5')pppAm- represents the capped and methylated 5'-end of mRNA: AdoMet + m7G(5')pppAm- leads to AdoHcy + m7G(5')pppm6A7- Greatest activity is obtained with RNA acceptors ending in m7G(5')pppAm-; less activity is found with RNA ending in m7G(5')pppA-; and barely detectable and no activity is found with RNA ending in G(5')pppA- and ppA-, respectively. Furthermore, no activity is found with oligonucleotides such as m7G(5')pppA, m7G(5')pppAm, and m7G(5')pppAmpN indicating that a longer polymer is required. It can be concluded from the substrate specificity of the enzyme that the formation of N6,2'-O-dimethyladenosine follows the biosynthesis of molecules containing m7G(5')pppAm-N-. The molecular weight of the methyltransferase, as determined by sedimentation in sucrose gradients, is approximately 65,000.

Adenosine↗
PubMed1983

Intramolecular stacking association and conformation properties of a 'cap' structure, m7G5'pppUm, and the related model compounds.

The stacking equilibrium quotient of the m7G5'pppUm unit, which occurs as the 5'-terminal "cap" of certain eukaryotic mRNA's, was determined by temperature-dependent difference spectrophotometry as Kstack = 1.82 at 25 degrees and pH 5. In order to evaluate the contribution of different structural modifications to the net stabilization of the cap structures of mRNA, a variety of compounds related to m7G5'pppUm were synthesized and their stacking properties were studied by the same method and compared. The results are summarized as: (1) Introduction of a methyl group into N-7 of G residue results in an increase in base stacking. (2) Methylation at 2'-OH of U residue also stabilizes the stacked structure of G-containing dimers, but it does not influence stacking interaction in m7G-containing dimers. (3) The effect of different types of internucleotide linkages on the order of stacking tendencies is: N5'ppN' greater than N5'pppN' greater than NpN'. UV hypochromicity and CD spectral measurements of the relevant dimers were also conducted, and the hypochromicity values and CD spectra of dimers in their stacked conformation were estimated by making use of the determined Kstack values. The results indicate that, while 2'-O-methylation exerts very little effect on the stacked conformation of the dimers, methylation at N-7 and the nature of the internucleotide linkage strongly influence the stacked conformation, thereby forming unusual left-handed conformations in m7G5'pppU(m), m7G5'ppU(m), and G5'ppU(m).

Nucleic Acid Conformation↗
PubMed1992

Trypanosoma brucei spliced-leader RNA methylations are required for trans splicing in vivo.

The Trypanosoma brucei spliced leader (SL) RNA donates its 5' leader sequence to all nuclear pre-mRNAs via trans RNA splicing. The SL RNA is a small-nuclear U RNA-like molecule which is present in the cell as part of a small ribonucleoprotein particle. However, unlike the trimethylguanosine-capped small nuclear U RNAs, the SL RNA has a highly modified 5' terminus containing an m7G cap and methylations on the first four transcribed nucleotides. Here, we show that incubation of procyclic-form T. brucei in the presence of the S-adenosylmethionine analog, sinefungin, leads to a rapid inhibition of SL RNA methylation. A concomitant inhibition of trans splicing and an accumulation of high-molecular-weight tubulin transcripts were also observed. The effects of sinefungin on SL RNA methylation and on trans splicing were correlated by labeling of cells incubated in the presence of the antibiotic. The results indicate that 5' modifications of the SL RNA are necessary for it to participate in trans splicing. SL RNA modification is not required for assembly of the core SL ribonucleoprotein, as these Cs2SO4-resistant particles can be formed with either methylated or undermethylated SL RNA.

Adenosine↗
PubMed1979

Nucleotide sequence of starfish initiator tRNA.

The nucleotide sequence of starfish ovary initiator tRNA was determined to be pA-G-C-A-G-A-G-U-m1G-m2G-C-G-C-A-G-U-G-G-A-A-G-C-G-U-G-C-U-G-G-G-C-C-C-A-U-t6A-A-C-C-C-A-G-A-G-m7G-D-m5C-C-G-A-G-G-A-psi-C-G-m1A-A-A-C-C-U-C-G-C-U-C-U-G-C-U-A-C-C-AOH. The sequence was determined by a combination of the two different post-labeling techniques. Two-dimensional cellulose thin-layer chromatography was adopted for analysis of 5'-terminal nucleotides of tRNA fragments produced by formamide treatment. The nucleotide sequence of starfish initiator tRNA is very similar to that of mammalian cytoplasmic initiator tRNAs, but has seven different nucleotide residues and two modifications: residue 55 is psi instead of U, and residue 26 is unmodified G instead of m2G.

Amino Acyl-tRNA Synthetases↗
PubMed1988

A detailed model of the three-dimensional structure of Escherichia coli 16 S ribosomal RNA in situ in the 30 S subunit.

A large body of intra-RNA and RNA-protein crosslinking data, obtained in this laboratory, was used to fold the phylogenetically and experimentally established secondary structure of Escherichia coli 16 S RNA into a three-dimensional model. All the crosslinks were induced in intact 30 S subunits (or in some cases in growing E. coli cells), and the sites of crosslinking were precisely localized on the RNA by oligonucleotide analysis. The RNA-protein crosslinking data (including 28 sites, and involving 13 of the 21 30S ribosomal were used to relate the RNA structure to the distribution of the proteins as determined by neutron scattering. The three-dimensional model of the 16 S RNA has overall dimensions of 220 A x 140 A x 90 A, in good agreement with electron microscopic estimates for the 30 S subunit. The shape of the model is also recognizably the same as that seen in electron micrographs, and the positions in the model of bases localized on the 30 S subunit by immunoelectron microscopy (the 5' and 3' termini, the m7G and m6(2)A residues, and C-1400) correspond closely to their experimentally observed positions. The distances between the RNA-protein crosslink sites in the model correlate well with the distances between protein centres of mass obtained by neutron scattering, only two out of 66 distances falling outside the expected tolerance limits. These two distances both involve protein S13, a protein noted for its anomalous behaviour. A comparison with other experimental information not specifically used in deriving the model shows that it fits well with published data on RNA-protein binding sites, mutation sites on the RNA causing resistance to antibiotics, tertiary interactions in the RNA, and a potential secondary structural "switch". Of the sites on 16 S RNA that have been found to be accessible to chemical modification in the 30 S subunit, 87% are at obviously exposed positions in the model. In contrast, 70% of the sites corresponding to positions that have ribose 2'-O-methylations in the eukaryotic 18 S RNA from Xenopus laevis are at non-exposed (i.e. internal) positions in the model. All nine of the modified bases in the E. coli 16 S RNA itself show a remarkable distribution, in that they form a "necklace" in one plane around the "throat" of the subunit. Insertions in eukaryotic 18 S RNA, and corresponding deletions in chloroplast or mammalian mitochondrial ribosomal RNA relative to E. coli 16 S RNA represent distinct sub-domains in the structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Computer Graphics↗
PubMed1999

Role of post-transcriptional modifications of primer tRNALys,3 in the fidelity and efficacy of plus strand DNA transfer during HIV-1 reverse transcription.

During HIV reverse transcription, (+) strand DNA synthesis is primed by an RNase H-resistant sequence, the polypurine tract, and continues as far as a 18-nt double-stranded RNA region corresponding to the 3' end of tRNALys,3 hybridized to the viral primer binding site (PBS). Before (+) strand DNA transfer, reverse transcriptase (RT) needs to unwind the double-stranded tRNA-PBS RNA in order to reverse-transcribe the 3' end of primer tRNALys,3. Since the detailed mechanism of (+) strand DNA transfer remains incompletely understood, we developed an in vitro system to closely examine this mechanism, composed of HIV 5' RNA, natural modified tRNALys,3, synthetic unmodified tRNALys,3 or oligonucleotides (RNA or DNA) complementary to the PBS, as well as the viral proteins RT and nucleocapsid protein (NCp7). Prior to (+) strand DNA transfer, RT stalls at the double-stranded tRNA-PBS RNA complex and is able to reverse-transcribe modified nucleosides of natural tRNALys,3. Modified nucleoside m1A-58 of natural tRNALys,3 is only partially effective as a stop signal, as RT can transcribe as far as the hyper-modified adenosine (ms2t6A-37) in the anticodon loop. m1A-58 is almost always transcribed into A, whereas other modified nucleosides are transcribed correctly, except for m7G-46, which is sometimes transcribed into T. In contrast, synthetic tRNALys,3, an RNA PBS primer, and a DNA PBS primer are completely reverse-transcribed. In the presence of an acceptor template, (+) strand DNA transfer is efficient only with templates containing natural tRNALys,3 or the RNA PBS primer. Sequence analysis of transfer products revealed frequent errors at the transfer site with synthetic tRNALys,3, not observed with natural tRNALys,3. Thus, modified nucleoside m1A-58, present in all retroviral tRNA primers, appears to be important for both efficacy and fidelity of (+) strand DNA transfer. We show that other factors such as the nature of the (-) PBS of the acceptor template and the RNase H activity of RT also influence the efficacy of (+) strand DNA transfer.

Base Sequence↗