Anticodon Loop Of tRNA Research Articles

Conformational Preferences of Modified Nucleoside N(4)-Acetylcytidine, ac4C Occur at “Wobble” 34th Position in the Anticodon Loop of tRNA

Conformational preferences of modified nucleoside, N(4)-acetylcytidine, ac(4)C have been investigated using quantum chemical semi-empirical RM1 method. Automated geometry optimization using PM3 method along with ab initio methods HF SCF (6-31G**), and density functional theory (DFT; B3LYP/6-31G**) have also been made to compare the salient features. The most stable conformation of N(4)-acetyl group of ac(4)C prefers "proximal" orientation. This conformation is stabilized by intramolecular hydrogen bonding between O(7)···HC(5), O(2)···HC2', and O4'···HC(6). The "proximal" conformation of N(4)-acetyl group has also been observed in another conformational study of anticodon loop of E. coli elongator tRNA(Met). The solvent accessible surface area (SASA) calculations revealed the role of ac(4)C in anticodon loop. The explicit molecular dynamics simulation study also shows the "proximal" orientation of N(4)-acetyl group. The predicted "proximal" conformation would allow ac(4)C to interact with third base of codon AUG/AUA whereas the 'distal' orientation of N(4)-acetyl cytidine side-chain prevents such interactions. Single point energy calculation studies of various models of anticodon-codon bases revealed that the models ac(4)C(34)(Proximal):G3, and ac(4)C(34)(Proximal):A3 are energetically more stable as compared to models ac(4)C(34)(Distal):G3, and ac(4)C(34)(Distal):A3, respectively. MEPs calculations showed the unique potential tunnels between the hydrogen bond donor-acceptor atoms of ac(4)C(34)(Proximal):G3/A3 base pairs suggesting role of ac(4)C in recognition of third letter of codons AUG/AUA. The "distal" conformation of ac(4)C might prevent misreading of AUA codon. Hence, this study could be useful to understand the role of ac(4)C in the tertiary structure folding of tRNA as well as in the proper recognition of codons during protein biosynthesis process.

1H, 13C and 15N resonance assignment of the N-terminal domain of human lysyl aminoacyl tRNA synthetase

Human lysyl aminoacyl tRNA synthetase (hLysRS) is integral to a variety of different functions ranging from protein biosynthesis, initiation of a proinflammatory response as well as signal transduction. Another important, non-canonical function of hLysRS is that it chaperones tRNA(Lys,3), the HIV-1 reverse transcription primer molecule into new HIV-1 particles. Since the N-terminal domain of hLysRS has been shown to be essential for such primer uptake, NMR studies of this domain are being conducted to obtain a better understanding of how hLysRS interacts with the primer tRNA. In order to study the RNA binding behavior of this domain, we are studying its complex with a fragment of the cognate tRNA corresponding to the tRNA anticodon loop. We report herein the backbone and side chain NMR resonance assignments of uniformly (15)N-, (13)C-labeled hLysRS N-terminal domain alone, as well as complexed to RNA.

Yeast Trm7 interacts with distinct proteins for critical modifications of the tRNAPhe anticodon loop

Post-transcriptional modification of the tRNA anticodon loop is critical for translation. Yeast Trm7 is required for 2'-O-methylation of C(32) and N(34) of tRNA(Phe), tRNA(Trp), and tRNA(Leu(UAA)) to form Cm(32) and Nm(34), and trm7-Δ mutants have severe growth and translation defects, but the reasons for these defects are not known. We show here that overproduction of tRNA(Phe) suppresses the growth defect of trm7-Δ mutants, suggesting that the crucial biological role of Trm7 is the modification of tRNA(Phe). We also provide in vivo and in vitro evidence that Trm7 interacts with ORF YMR259c (now named Trm732) for 2'-O-methylation of C(32), and with Rtt10 (named Trm734) for 2'-O-methylation of N(34) of substrate tRNAs and provide evidence for a complex circuitry of anticodon loop modification of tRNA(Phe), in which formation of Cm(32) and Gm(34) drives modification of m(1)G(37) (1-methylguanosine) to yW (wyebutosine). Further genetic analysis shows that the slow growth of trm7-Δ mutants is due to the lack of both Cm(32) and Nm(34), and the accompanying loss of yW, because trm732-Δ trm734-Δ mutants phenocopy trm7-Δ mutants, whereas each single mutant is healthy; nonetheless, TRM732 and TRM734 each have distinct roles, since mutations in these genes have different genetic interactions with trm1-Δ mutants, which lack m(2,2)G(26) in their tRNAs. We speculate that 2'-O-methylation of the anticodon loop may be important throughout eukaryotes because of the widespread conservation of Trm7, Trm732, and Trm734 proteins, and the corresponding modifications, and because the putative human TRM7 ortholog FTSJ1 is implicated in nonsyndromic X-linked mental retardation.

Mapping the tRNA Binding Site on the Surface of Human DNMT2 Methyltransferase

The DNMT2 enzyme methylates tRNA-Asp at position C38. Because there is no tRNA-Dnmt2 cocrystal structure available, we have mapped the tRNA binding site of DNMT2 by systematically mutating surface-exposed lysine and arginine residues to alanine and studying the tRNA methylation activity and binding of the corresponding variants. After mutating 20 lysine and arginine residues, we identified eight of them that caused large (>4-fold) decreases in catalytic activity. These residues cluster within and next to a surface cleft in the protein, which is large enough to accommodate the tRNA anticodon loop and stem. This cleft is located next to the binding pocket for the cofactor S-adenosyl-L-methionine, and the catalytic residues of DNMT2 are positioned at its walls or bottom. Many of the variants with strongly reduced catalytic activity showed only a weak loss of tRNA binding or even bound better to tRNA than wild-type DNMT2, which suggests that the enzyme induces some conformational changes in the tRNA in the transition state of the methyl group transfer reaction. Manual placement of tRNA into the structure suggests that DNMT2 mainly interacts with the anticodon stem and loop.

Solution Nuclear Magnetic Resonance Analyses of the Anticodon Arms of Proteinogenic and Nonproteinogenic tRNAGly

Although the fate of most tRNA molecules in the cell is aminoacylation and delivery to the ribosome, some tRNAs are destined to fulfill other functional roles. In addition to their central role in translation, tRNA molecules participate in processes such as regulation of gene expression, bacterial cell wall biosynthesis, viral replication, antibiotic biosynthesis, and suppression of alternative splicing. In bacteria, glycyl-tRNA molecules with anticodon sequences GCC and UCC exhibit multiple extratranslational functions, including transcriptional regulation and cell wall biosynthesis. We have determined the high-resolution structures of three glycyl-tRNA anticodon arms with anticodon sequences GCC and UCC. Two of the tRNA molecules are proteinogenic (tRNA(Gly,GCC) and tRNA(Gly,UCC)), and the third is nonproteinogenic (np-tRNA(Gly,UCC)) and participates in cell wall biosynthesis. The UV-monitored thermal melting curves show that the anticodon arm of tRNA(Gly,UCC) with a loop-closing C-A(+) base pair melts at a temperature 10 °C lower than those of tRNA(Gly,GCC) and np-tRNA(Gly,UCC). U-A and C-G pairs close the loops of the latter two molecules and enhance stem stability. Mg(2+) stabilizes the tRNA(Gly,UCC) anticodon arm and reduces the T(m) differential. The structures of the three tRNA(Gly) anticodon arms exhibit small differences among one another, but none of them form the classical U-turn motif. The anticodon loop of tRNA(Gly,GCC) becomes more dynamic and disordered in the presence of multivalent cations, whereas metal ion coordination in the anticodon loops of tRNA(Gly,UCC) and np-tRNA(Gly,UCC) establishes conformational homogeneity. The conformational similarity of the molecules is greater than their functional differences might suggest. Because aminoacylation of full-length tRNA molecules is accomplished by one tRNA synthetase, the similar structural context of the loop may facilitate efficient recognition of each of the anticodon sequences.

Identification of a target cell permissive factor required for contact-dependent growth inhibition (CDI).

Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CdiA effector proteins are exported onto the surface of CDI(+) inhibitor cells, where they interact with susceptible bacteria and deliver effectors/toxins derived from their C-terminal regions (CdiA-CT). CDI(+) cells also produce an immunity protein that binds the CdiA-CT and blocks its activity to prevent autoinhibition. Here, we show that the CdiA-CT from uropathogenic Escherichia coli strain 536 (UPEC536) is a latent tRNase that requires activation by the biosynthetic enzyme CysK (O-acetylserine sulfhydrylase A). UPEC536 CdiA-CT exhibits no nuclease activity in vitro, but cleaves within transfer RNA (tRNA) anti-codon loops when purified CysK is added. CysK and CdiA-CT form a stable complex, and their binding interaction appears to mimic that of the CysK/CysE cysteine synthase complex. CdiA-CT activation is also required for growth inhibition. Synthesis of CdiA-CT in E. coli cysK(+) cells arrests cell growth, whereas the growth of ΔcysK mutants is unaffected by the toxin. Moreover, E. coli ΔcysK cells are completely resistant to inhibitor cells expressing UPEC536 CdiA, indicating that CysK is required to activate the tRNase during CDI. Thus, CysK acts as a permissive factor for CDI, providing a potential mechanism to modulate growth inhibition in target cells.

Amino acid–anticodon binding specificity: rationale for a new class of therapeutic agent

In this article a new class of anticancer and antiviral drugs is discussed. These new drugs consist of small di- and tri-peptides, designed to bind to single-stranded (ss) regions that are crucial for the expression of genes such as the c-myc oncogene in cancers and start sites (and other ss regions) of viral pathogenic genes. The components (i.e. the amino acids and the sequences they form) of these peptides could be dictated by the specific binding of amino acids to their ss anticodons in tRNA. Cancer cell viability depends on the continued overexpression of the c-myc oncogene, and thus this gene is a target of opportunity for anticancer agents. Sharply reducing the overexpression of c-myc leads to the death of cancer cells. To achieve this end the following rationale is suggested: crucial regions of the c-myc promoters (to which activating proteins must bind for expression to occur) are single stranded and thus strongly resemble the anticodon loop of tRNA. It was found that amino acids chemically bind to their cognate tRNA anticodons. Regarding the ss regions of c-myc as a series of adjacent 'anticodons', di- and tri-peptides are proposed to be aligned to their cognate 'anticodons' in the proper order. For example, if the ss region of a promoter is hypothetically TTT-GGG-CCC, the tripeptide Lys-Pro-Gly could be expected to bind to it and deny access of the promoter to all activating proteins, thereby blocking c-myc expression and all the cancers dependent on such overexpression. Similarly, it is reported that in the initial phase of gene expression the start sites of the genes are single stranded (before and after and spanning the start site). Thus, invoking the amino acid cognate anticodon binding specificity (ACABS) principle as described above, a series of small peptides are suggested that could span the start sites of pathogenic viral genes (e.g. the oris region of herpes simplex virus (HSV)) to deny access of the gene to the transcription elements. This would inactivate the toxic effect of the virus and thereby constitute a promising approach to antiviral therapy, where the start sites (or other ss regions of pathogenic genes) have been sequenced. The ACABS principle (for peptide-nucleic-acid interaction) enables us to focus on probable effective small peptides rather than having to screen a large number of randomly chosen small peptides to find probable anticancer and antiviral therapeutic agents.

A domain of the actin binding protein Abp140 is the yeast methyltransferase responsible for 3-methylcytidine modification in the tRNA anti-codon loop

The 3-methylcytidine (m³C) modification is widely found in eukaryotic species of tRNA(Ser), tRNA(Thr), and tRNA(Arg); at residue 32 in the anti-codon loop; and at residue e2 in the variable stem of tRNA(Ser). Little is known about the function of this modification or about the specificity of the corresponding methyltransferase, since the gene has not been identified. We have used a primer extension assay to screen a battery of methyltransferase candidate knockout strains in the yeast Saccharomyces cerevisiae, and find that tRNA(Thr(IGU)) from abp140-Δ strains lacks m³C. Curiously, Abp140p is composed of a poorly conserved N-terminal ORF fused by a programed +1 frameshift in budding yeasts to a C-terminal ORF containing an S-adenosylmethionine (SAM) domain that is highly conserved among eukaryotes. We show that ABP140 is required for m³C modification of substrate tRNAs, since primer extension is similarly affected for all tRNA species expected to have m³C and since quantitative analysis shows explicitly that tRNA(Thr(IGU)) from an abp140-Δ strain lacks m³C. We also show that Abp140p (now named Trm140p) purified after expression in yeast or Escherichia coli has m³C methyltransferase activity, which is specific for tRNA(Thr(IGU)) and not tRNA(Phe) and occurs specifically at C₃₂. We suggest that the C-terminal ORF of Trm140p is necessary and sufficient for activity in vivo and in vitro, based on analysis of constructs deleted for most or all of the N-terminal ORF. We also suggest that m³C has a role in translation, since trm140-Δ trm1-Δ strains (also lacking m²,²G₂₆) are sensitive to low concentrations of cycloheximide.

Solution Structure of the K-Turn and Specifier Loop Domains from the Bacillus subtilis tyrS T-Box Leader RNA

In Gram-positive bacteria, the RNA transcripts of many amino acid biosynthetic and aminoacyl tRNA synthetase genes contain 5′ untranslated regions, or leader RNAs, that function as riboswitches. These T-box riboswitches bind cognate tRNA molecules and regulate gene expression by a transcription attenuation mechanism. The Specifier Loop domain of the leader RNA contains nucleotides that pair with nucleotides in the tRNA anticodon loop and is flanked on one side by a kink-turn (K-turn), or GA, sequence motif. We have determined the solution NMR structure of the K-turn sequence element within the context of the Specifier Loop domain. The K-turn sequence motif has several noncanonical base pairs typical of K-turn structures but adopts an extended conformation. The Specifier Loop domain contains a loop E structural motif, and the single-strand Specifier nucleotides stack with their Watson–Crick edges displaced toward the minor groove. Mg2+ leads to a significant bending of the helix axis at the base of the Specifier Loop domain, but does not alter the K-turn. Isothermal titration calorimetry indicates that the K-turn sequence causes a small enhancement of the interaction between the tRNA anticodon arm and the Specifier Loop domain. One possibility is that the K-turn structure is formed and stabilized when tRNA binds the T-box riboswitch and interacts with Stem I and the antiterminator helix. This motif in turn anchors the orientation of Stem I relative to the 3′ half of the leader RNA, further stabilizing the tRNA–T box complex.

Idiosyncrasy and identity in the prokaryotic phe‐system: Crystal structure of E. coli phenylalanyl‐tRNA synthetase complexed with phenylalanine and AMP

The crystal structure of Phenylalanyl-tRNA synthetase from E. coli (EcPheRS), a class II aminoacyl-tRNA synthetase, complexed with phenylalanine and AMP was determined at 3.05 Å resolution. EcPheRS is a (αβ)₂ heterotetramer: the αβ heterodimer of EcPheRS consists of 11 structural domains. Three of them: the N-terminus, A1 and A2 belong to the α-subunit and B1-B8 domains to the β subunit. The structure of EcPheRS revealed that architecture of four helix-bundle interface, characteristic of class IIc heterotetrameric aaRSs, is changed: each of the two long helices belonging to CLM transformed into the coil-short helix structural fragments. The N-terminal domain of the α-subunit in EcPheRS forms compact triple helix domain. This observation is contradictory to the structure of the apo form of TtPheRS, where N-terminal domain was not detected in the electron density map. Comparison of EcPheRS structure with TtPheRS has uncovered significant rearrangements of the structural domains involved in tRNA(Phe) binding/translocation. As it follows from modeling experiments, to achieve a tighter fit with anticodon loop of tRNA, a shift of ∼5 Å is required for C-terminal domain B8, and of ∼6 to 7 Å for the whole N terminus. EcPheRSs have emerged as an important target for the incorporation of novel amino acids into genetic code. Further progress in design of novel compounds is anticipated based on the structural data of EcPheRS.

The Human Mitochondrial tRNA Met: Structure/Function Relationship of a Unique Modification in the Decoding of Unconventional Codons

Human mitochondrial mRNAs utilize the universal AUG and the unconventional isoleucine AUA codons for methionine. In contrast to translation in the cytoplasm, human mitochondria use one tRNA, hmtRNA Met CAU, to read AUG and AUA codons at both the peptidyl- (P-), and aminoacyl- (A-) sites of the ribosome. The hmtRNA Met CAU has a unique post-transcriptional modification, 5-formylcytidine, at the wobble position 34 (f 5C 34), and a cytidine substituting for the invariant uridine at position 33 of the canonical U-turn in tRNAs. The structure of the tRNA anticodon stem and loop domain (hmtASL Met CAU), determined by NMR restrained molecular modeling, revealed how the f 5C 34 modification facilitates the decoding of AUA at the P- and the A-sites. The f 5C 34 defined a reduced conformational space for the nucleoside, in what appears to have restricted the conformational dynamics of the anticodon bases of the modified hmtASL Met CAU. The hmtASL Met CAU exhibited a C-turn conformation that has some characteristics of the U-turn motif. Codon binding studies with both Escherichia coli and bovine mitochondrial ribosomes revealed that the f 5C 34 facilitates AUA binding in the A-site and suggested that the modification favorably alters the ASL binding kinetics. Mitochondrial translation by many organisms, including humans, sometimes initiates with the universal isoleucine codons AUU and AUC. The f 5C 34 enabled P-site codon binding to these normally isoleucine codons. Thus, the physicochemical properties of this one modification, f 5C 34, expand codon recognition from the traditional AUG to the non-traditional, synonymous codons AUU and AUC as well as AUA, in the reassignment of universal codons in the mitochondria.

Structural rearrangements of the ribosome at the tRNA proofreading step

Discrimination of tRNA on the ribosome occurs in two consecutive steps: initial selection and proofreading. Here we propose a proofreading mechanism based on comparison of crystal structures of the 70S ribosome with an empty A site or with the A site occupied by uncharged cognate or near-cognate tRNA. We observe that ribosomal proteins S13, S19, L16, L25, L27 and L31 are actively involved in the proofreading of tRNA. We suggest that proofreading begins with the monitoring of the entire anticodon loop of tRNA by nucleotides from 16S rRNA (helices 18 and 44) of the small subunit and 23S rRNA (helix 69) of the large subunit with involvement of magnesium ions. Subsequently, the elbow region is scanned by rRNA (helices 38 and 89) and proteins from the large subunit determining whether to accommodate the acceptor end of tRNA in the peptidyl transferase center or not.

NMR structure and dynamics of the Specifier Loop domain from the Bacillus subtilis tyrS T box leader RNA

Gram-positive bacteria utilize a tRNA-responsive transcription antitermination mechanism, designated the T box system, to regulate expression of many amino acid biosynthetic and aminoacyl-tRNA synthetase genes. The RNA transcripts of genes controlled by this mechanism contain 5′ untranslated regions, or leader RNAs, that specifically bind cognate tRNA molecules through pairing of nucleotides in the tRNA anticodon loop with nucleotides in the Specifier Loop domain of the leader RNA. We have determined the solution structure of the Specifier Loop domain of the tyrS leader RNA from Bacillus subtilis. Fifty percent of the nucleotides in the Specifier Loop domain adopt a loop E motif. The Specifier Sequence nucleotides, which pair with the tRNA anticodon, stack with their Watson–Crick edges rotated toward the minor groove and exhibit only modest flexibility. We also show that a Specifier Loop domain mutation that impairs the function of the B. subtilis glyQS T box RNA disrupts the tyrS loop E motif. Our results suggest a mechanism for tRNA–Specifier Loop binding in which the phosphate backbone kink created by the loop E motif causes the Specifier Sequence bases to rotate toward the minor groove, which increases accessibility for pairing with bases in the anticodon loop of tRNA.

Mitochondrial tRNAs as light strand replication origins: Similarity between anticodon loops and the loop of the light strand replication origin predicts initiation of DNA replication

Stem-loop hairpins formed by mitochondrial light strand replication origins (OL) and by heavy strand DNA coding for tRNAs that form OL-like structures initiate mitochondrial replication. The loops are recognized by one of the two active sites of the vertebrate mitochondrial gamma polymerase, which are homologous to the active sites of class II amino-acyl tRNA synthetases. Therefore, the polymerase site recognizing the OL loop could recognize tRNA anticodon loops and sequence similarity between anticodon and OL loops should predict initiation of DNA replication at tRNAs. Strengths of genome-wide deamination gradients starting at tRNA genes estimate extents by which replication starts at that tRNA. Deaminations (A-->G and C-->T) occur proportionally to time spent single stranded by heavy strand DNA during mitochondrial light strand replication. Results show that deamination gradients starting at tRNAs are proportional to sequence similarity between OL and tRNA loops: most for anticodon-, least D-, intermediate for TpsiC-loops, paralleling tRNA synthetase recognition interactions with these tRNA loops. Structural and sequence similarities with regular OLs predict OL function, loop similarity is dominant in most tRNAs. Analyses of sequence similarity and structure independently substantiate that DNA sequences coding for mitochondrial tRNAs sometimes function as alternative OLs. Pathogenic mutations in anticodon loops increase similarity with the human OL loop, non-pathogenic polymorphisms do not. Similarity/homology alignment hypotheses are experimentally testable in this system.

Bases in the anticodon loop of tRNAAlaGGC prevent misreading

The bases at positions 32 and 38 in the tRNA anticodon loop are known to have a specific conservation depending upon the anticodon triplets. Here we report that evolutionarily conserved pairs of bases at positions 32 and 38 in tRNA(Ala)(GGC) prevent misreading of a near-cognate valine codon, GUC. The tRNA(Ala)(GGC) molecules with the conserved A32-U38 and C32-G38 pairs do not read GUC, whereas those with three representative nonconserved pairs, U32-U38, U32-A38 and C32-A38, direct the misincorporation of alanine at this valine codon into the peptide chain. Overexpression of the nonconserved tRNA(Ala)(GGC) in Escherichia coli is toxic and prevents cell growth. These results suggested that the bases at positions 32 and 38 in tRNA(Ala)(GGC) evolved to preserve the fidelity of the cognate codon reading.

RNA-Protein Mutually Induced Fit

tRNAs that read codons starting with U are usually modified at their A37 by isopentenyl-tRNA transferases to minimize peptidyl-tRNA slippage in translation. The consensus substrate requirements of the isopentenyl-tRNA transferase of Escherichia coli, MiaA, have been the focus of extensive study. However, the molecular basis of tRNA-MiaA recognition remains unknown. Here we describe the 2.5A crystal structure of MiaA in complex with substrate tRNA(Phe). Comparative structural analysis reveals that the enzymatic reaction involves an RNA-protein mutually induced fit mechanism in which large domain movements in MiaA provoke the partial unfolding of the substrate tRNA anticodon loop. In addition, we show how substrate tRNAs are recognized by MiaA through a combination of direct and indirect sequence readouts.

A gripping tale of ribosomal frameshifting: extragenic suppressors of frameshift mutations spotlight P-site realignment.

Mutants of translation components which compensate for both -1 and +1 frameshift mutations showed the first evidence for framing malleability. Those compensatory mutants isolated in bacteria and yeast with altered tRNA or protein factors are reviewed here and are considered to primarily cause altered P-site realignment and not altered translocation. Though the first sequenced tRNA mutant which suppressed a +1 frameshift mutation had an extra base in its anticodon loop and led to a textbook "yardstick" model in which the number of anticodon bases determines codon size, this model has long been discounted, although not by all. Accordingly, the reviewed data suggest that reading frame maintenance and translocation are two distinct features of the ribosome. None of the -1 tRNA suppressors have anticodon loops with fewer than the standard seven nucleotides. Many of the tRNA mutants potentially affect tRNA bending and/or stability and can be used for functional assays, and one has the conserved C74 of the 3' CCA substituted. The effect of tRNA modification deficiencies on framing has been particularly informative. The properties of some mutants suggest the use of alternative tRNA anticodon loop stack conformations by individual tRNAs in one translation cycle. The mutant proteins range from defective release factors with delayed decoding of A-site stop codons facilitating P-site frameshifting to altered EF-Tu/EF1alpha to mutant ribosomal large- and small-subunit proteins L9 and S9. Their study is revealing how mRNA slippage is restrained except where it is programmed to occur and be utilized.

Molecular Dynamics Study of the Ribosomal A-Site

Many aminoglycosidic antibiotics target the A-site of 16S RNA in the small ribosomal subunit and affect the fidelity of protein translation in bacteria. Upon binding, aminoglycosides displace two adenines (A1492 and A1493 for E. coli numbering) that are involved in tRNA anticodon loop recognition. The major difference in the aminoglycosidic binding site between the prokaryota and eukaryota is an adenine into guanine substitution in the position 1408. This mutation likely affects the dynamics of near A1492 and A1493 and hinders the binding of aminoglycosides to eukaryotic ribosomes. With multiple 20 ns long all-atom molecular dynamics simulations, we study the flexibility of a 22 nucleotide RNA fragment which mimics the aminoglycosidic binding site. Simulations are carried out for both native and A1408G mutated RNA as well as for their complexes with aminoglycosidic representative paromomycin. We observe intra- and extrahelical configurations of A1492 and A1493, which differ between the prokaryotic and the mutated structure. We obtain configurations of the A-site that are also observed in the NMR and crystal structures. Our studies show the differences in the internal mobility of the A-site, as well as that in ion and water density distributions inside of the binding cleft, between the prokaryotic and mutated RNA. We also compare the performance of two force field parameters for RNA, Amber and Charmm.

Crystallographic snapshots of eukaryotic dimethylallyltransferase acting on tRNA: insight into tRNA recognition and reaction mechanism.

Hypermodifications near the anticodon of tRNA are fundamental for the efficiency and fidelity of protein synthesis. Dimethylallyltransferase (DMATase) catalyzes transfer of a dimethylallyl moiety from dimethylallyl pyrophosphate to N6 of A37 in certain tRNAs. Here we present the crystal structures of Saccharomyces cerevisiae DMATase-tRNA(Cys) complex in four distinct forms, which provide snapshots of the RNA modification reaction catalyzed by DMATase. The structures reveal that the enzyme recognizes the tRNA substrate through indirect sequence readout. The targeted nucleotide A37 flips out from the anticodon loop of tRNA and flips into a channel in DMATase, where it meets its reaction partner di methylallyl pyrophosphate, which enters the channel from the opposite end. Structural changes accompanying the transfer reaction taking place in the crystal result in disengagement of DMATase-tRNA interaction near the reaction center. In addition, structural comparison of DMATase in the complex with unliganded bacterial DMATase provides a molecular basis of ordered substrate binding by DMATase.

Anticodon loop mutations perturb reading frame maintenance by the E site tRNA

The ribosomal E site helps hold the reading frame. Certain tRNA mutations affect translation, and anticodon loop mutations can be especially detrimental. We studied the effects of mutations saturating the anticodon loop of the amber suppressor tRNA, Su7, on the ability to help hold the reading frame when in the E site. We also tested three mutations in the anticodon stem, as well as a mutation in the D stem (the "Hirsh" mutation). We used the Escherichia coli RF2 programmed frameshift site to monitor frame maintenance. Most anticodon loop mutations increase frameshifting, possibly by decreasing codon:anticodon stability. However, it is likely that the A site is more sensitive to anticodon loop structure than is the E site. Unexpectedly, the Hirsh mutation also increases frameshifting from the E site. Other work shows that mutation may increase the ability of tRNA to react in the A site, possibly by facilitating conformational changes required for aminoacyl-tRNA selection. We suggest that this property may decrease its ability to bind to the E site. Finally, the absence of the ms(2)io(6)A nucleoside modifications at A37 does not decrease the ability of tRNA to help hold the reading frame from the E site. This was also unexpected because the absence of these modifications affects translational properties of tRNA in A and P sites. The absence of a negative effect in the E site further highlights the differences among the substrate requirements of the ribosomal coding sites.