Role Of tRNA Research Articles

HIV‐1 Matrix Protein Interactions with Cellular tRNAs

The human immunodeficiency virus (HIV) is a retrovirus that infects roughly 40 million people globally that attacks CD4+ T‐cells, thus impairing the immune system. Highly active antiretroviral treatment against the virus is available; however, the viral genome frequently mutates, which gives rise to evasion of current therapies and highlights the need for new drugs. Better understanding molecular mechanisms of viral replication may lend to this effort. One process necessary for HIV replication is membrane trafficking of the Gag polyprotein (Gag) to the plasma membrane via Gag’s N‐terminal matrix domain (MA). MA mediates membrane targeting by means of electrostatic interaction and anchoring to the plasma membrane using a covalently linked myristoyl moiety. Recent virology studies demonstrated that MA interacts with cellular tRNAs, including tRNALys3 and tRNAGlyGCC, before binding to the plasma membrane, but the role of tRNA on assembly is poorly understood. The long‐term goal of this work is to study the MA:tRNA complex and elucidate its 3D structure as characterization of this interaction may shed light on the function of tRNA during membrane targeting. To achieve this goal, conditions to stabilize MA and the tRNA independently and in complex must be established. Nuclear magnetic resonance (NMR) and gel electrophoresis were employed to identify conditions that stabilized the tRNA and MA independently. Electrophoretic mobility shift assays (EMSAs) were carried out to probe MA:tRNA interactions under these conditions and showed complex formation upon addition of a stoichiometric ratio of MA to tRNA. This work lays a foundation to study thermodynamics of the MA‐tRNA interaction and determine the three‐dimensional structure of the complex.Support or Funding InformationThis research was sponsored by NIH/NIGMS MARC U*STAR T34 HHS 00026 National Research Service Award to UMBC, HHMI, and NIH grants R01GM042561 and 8R01AI50498.

The Role of tRNA in Establishing New Genetic Codes.

One of the most remarkable, but typically unremarked, aspects of the translation apparatus is the pleiotropic pliability of tRNA. This humble cloverleaf/L-shaped molecule must implement the first genetic code, via base pairing and wobble interactions, but is also largely responsible for the specificity of the second genetic code, the pairings between amino acids, tRNA synthetases, and tRNAs. Despite the overarching similarities between tRNAs, they must nonetheless be specifically recognized by cognate tRNA synthetases and largely rejected by noncognate synthetases. Conversely, despite the differences between tRNAs that allow such discrimination, they must be uniformly accepted by the ribosome, in part via the machinations of the translation elongation factors, which work with a diverse coterie of tRNA-amino acid conjugates to balance binding and loading. While it is easy to ascribe both discrimination and acceptance to the individual proteins (synthetases and EF-Tu/eEF-1) that recognize tRNAs, there is a large body of evidence that suggests that the sequences, structures, and dynamics of tRNAs are instrumental in the choices these proteins make.

Understanding the role of tRNA modifying enzymes in proteostasis in human health and disease – FCT

Understanding the role of tRNA modifying enzymes in proteostasis in human health and disease – FCT

Chance and necessity in the evolution of RNase P.

RNase P catalyzes 5′-maturation of tRNAs in all three domains of life. This primary function is accomplished by either a ribozyme-centered ribonucleoprotein (RNP) or a protein-only variant (with one to three polypeptides). The large, multicomponent archaeal and eukaryotic RNase P RNPs appear disproportionate to the simplicity of their role in tRNA 5′-maturation, prompting the question of why the seemingly gratuitously complex RNP forms of RNase P were not replaced with simpler protein counterparts. Here, motivated by growing evidence, we consider the hypothesis that the large RNase P RNP was retained as a direct consequence of multiple roles played by its components in processes that are not related to the canonical RNase P function.

The Escherichia coli COG1738 Member YhhQ Is Involved in 7-Cyanodeazaguanine (preQ₀) Transport.

Queuosine (Q) is a complex modification of the wobble base in tRNAs with GUN anticodons. The full Q biosynthesis pathway has been elucidated in Escherichia coli. FolE, QueD, QueE and QueC are involved in the conversion of guanosine triphosphate (GTP) to 7-cyano-7-deazaguanine (preQ0), an intermediate of increasing interest for its central role in tRNA and DNA modification and secondary metabolism. QueF then reduces preQ0 to 7-aminomethyl-7-deazaguanine (preQ1). PreQ1 is inserted into tRNAs by tRNA guanine(34) transglycosylase (TGT). The inserted base preQ1 is finally matured to Q by two additional steps involving QueA and QueG or QueH. Most Eubacteria harbor the full set of Q synthesis genes and are predicted to synthesize Q de novo. However, some bacteria only encode enzymes involved in the second half of the pathway downstream of preQ0 synthesis, including the signature enzyme TGT. Different patterns of distribution of the queF, tgt, queA and queG or queH genes are observed, suggesting preQ0, preQ1 or even the queuine base being salvaged in specific organisms. Such salvage pathways require the existence of specific 7-deazapurine transporters that have yet to be identified. The COG1738 family was identified as a candidate for a missing preQ0/preQ1 transporter in prokaryotes, by comparative genomics analyses. The existence of Q precursor salvage was confirmed for the first time in bacteria, in vivo, through an indirect assay. The involvement of the COG1738 in salvage of a Q precursor was experimentally validated in Escherichia coli, where it was shown that the COG1738 family member YhhQ is essential for preQ0 transport.

Molecular Recognition of tRNA with 1-Naphthyl Acetyl Spermine, Spermine, and Spermidine: A Thermodynamic, Biophysical, and Molecular Docking Investigative Approach.

The role of tRNA in protein translational machinery and the influence of polyamines on the interaction of acylated and deacylated tRNA with ribosomes make polyamine-tRNA interaction conspicuous. We studied the interaction of two biogenic polyamines, spermine (SPM) and spermidine (SPD), with tRNAPhe and compared the results to those of the analogue 1-naphthyl acetyl spermine (NASPM). The binding affinity of SPM was comparable to that of NASPM; both were higher than that of SPD. The interactions led to significant thermal stabilization of tRNAPhe and an increase in the enthalpy of transition. All the interactions were exothermic in nature and displayed prominent enthalpy-entropy compensation behavior. The entropy-driven nature of the interaction, the structural perturbations observed, and docking results proved that the polyamines were bound in the groove of the anticodon arm of tRNAPhe. The amine groups of polyamines were involved in extensive electrostatic, H-bonding, and van der Waals interactions with tRNAPhe. The naphthyl group of NASPM showed an additional stacking interaction with G24 and G26 of tRNAPhe, which was absent in others. The results demonstrate that 1-naphthyl acetyl spermine can target the same binding sites as the biogenic polyamines without substituting for the functions played by them, which may lead to exhibition of selective anticancer cytotoxicity.

The Diversity of Ribonuclease P: Protein and RNA Catalysts with Analogous Biological Functions.

Ribonuclease P (RNase P) is an essential endonuclease responsible for catalyzing 5’ end maturation in precursor transfer RNAs. Since its discovery in the 1970s, RNase P enzymes have been identified and studied throughout the three domains of life. Interestingly, RNase P is either RNA-based, with a catalytic RNA subunit, or a protein-only (PRORP) enzyme with differential evolutionary distribution. The available structural data, including the active site data, provides insight into catalysis and substrate recognition. The hydrolytic and kinetic mechanisms of the two forms of RNase P enzymes are similar, yet features unique to the RNA-based and PRORP enzymes are consistent with different evolutionary origins. The various RNase P enzymes, in addition to their primary role in tRNA 5’ maturation, catalyze cleavage of a variety of alternative substrates, indicating a diversification of RNase P function in vivo. The review concludes with a discussion of recent advances and interesting research directions in the field.

Theoretical Analysis of a Self-Replicator With Reduced Template Inhibition Based on an Informational Leaving Group.

The first non-enzymatic self-replicating systems, as proposed by von Kiedrowski (Angew Chem Int Ed Engl 25(10):932-935, 1986) and Orgel (Nature 327(6120):346-347, 1987), gave rise to the analytical background still used today to describe artificial replicators. What separates a self-replicating from an autocatalytic system is the ability to pass on structural information (Orgel, Nature 358(6383):203-209, 1992). Utilising molecular information, nucleic acids were the first choice as prototypical examples. But early self-replicators showed parabolic over exponential growth due to the strongly bound template duplex after template-directed ligation of substrates. We propose a self-replicating scheme with a weakly bound template duplex, using an informational leaving group. Such a scheme is inspired by the role of tRNA as leaving group and information carrier during protein synthesis, and is based on our previous experience with nucleotide chemistry. We analyse theoretically this scheme and compare it to the classical minimal replicator model. We show that for an example hexanucleotide template mirroring that is used by von Kiedrowski (Bioorganic chemistry frontiers, 1993) for the analysis of the classical minimal replicator, the proposed scheme is expected to result in higher template self-replication rate. The proposed self-replicating scheme based on an informational leaving group is expected to outperform the classical minimal replicator because of a weaker template duplex bonding, resulting in reduced template inhibition.

Synergistic effects of ATP and RNA binding to human DEAD-box protein DDX1.

RNA helicases of the DEAD-box protein family form the largest group of helicases. The human DEAD-box protein 1 (DDX1) plays an important role in tRNA and mRNA processing, is involved in tumor progression and is also hijacked by several virus families such as HIV-1 for replication and nuclear export. Although important in many cellular processes, the mechanism of DDX1′s enzymatic function is unknown. We have performed equilibrium titrations and transient kinetics to determine affinities for nucleotides and RNA. We find an exceptional tight binding of DDX1 to adenosine diphosphate (ADP), one of the strongest affinities observed for DEAD-box helicases. ADP binds tighter by three orders of magnitude when compared to adenosine triphosphate (ATP), arresting the enzyme in a potential dead-end ADP conformation under physiological conditions. We thus suggest that a nucleotide exchange factor leads to DDX1 recycling. Furthermore, we find a strong cooperativity in binding of RNA and ATP to DDX1 that is also reflected in ATP hydrolysis. We present a model in which either ATP or RNA binding alone can partially shift the equilibrium from an ‘open’ to a ‘closed’-state; this shift appears to be not further pronounced substantially even in the presence of both RNA and ATP as the low rate of ATP hydrolysis does not change.

The natural history of transfer RNA and its interactions with the ribosome.

Transfer RNA (tRNA) is undoubtedly the most central and one of the oldest molecules of the cell. Without it genetics and coded protein synthesis are impossible. The crucial specificities responsible for the genetic code and accurate translation are by far entrusted to interactions between tRNA and translation proteins, fundamentally aminoacyl-tRNA synthetase (aaRS) enzymes and elongation factor (EF) switches (Yadavalli and Ibba, 2012). Discrimination mediated by aaRSs and EFs against misincorporated tRNA and amino acids is at least 20 times more stringent than ribosomal recognition, editing, and other proofreading mechanisms (Reynolds et al., 2010). The fact that crucial genetic code specificities in highly selective interactions with protein enzymes do not involve the ribosomal ribonucleoprotein biosynthetic machinery challenges the “replicators first” origin of life scenario of an ancient RNA world (Caetano-Anolles and Seufferheld, 2013). It also highlights the central functional, mechanistic, and evolutionary roles of tRNA and its recognition determinants, which enable coevolution between nucleic acids and proteins. These coevolutionary relationships are compatible with a late origin of the ribosome in its mechanism and not in protein biosynthesis, which was inferred from the computational analysis of thousands of RNAs and proteomes (Harish and Caetano-Anolles, 2012). These analyses showed tight coevolution of ribosomal RNA (rRNA) and ribosomal proteins (r-proteins). While these relationships delimit molecular makeup when organisms use translation to negotiate growth and viability amidst environmental change, coevolution also constrains recruitment of the canonical L-shaped structure of the tRNA molecule into a multiplicity of modern functions. These new functions include the synthesis of antibiotics, bacterial cell wall peptidoglycans and tetrapyrroles, modification of bacterial membrane lipids, protein turnover, and the synthesis of other aminoacyl-tRNA molecules (Francklyn and Minajigi, 2010). Here we unfold coevolutionary relationships between tRNA substructures and translation proteins that embody crucial protein-nucleic acid interactions. We focus on a series of computational biology analyses of the structure and conformational diversity of tRNAs and their interacting proteins that provide information about the history of structural accretion of this “adaptor” molecule. Using this information, we place tRNA history within the framework of an evolutionary timeline of protein domain innovation, uncovering the natural history of tRNA within the context of the geological record.

A conserved RNA polymerase III promoter required for gammaherpesvirus TMER transcription and microRNA processing.

Canonical RNA polymerase III (pol III) type 2 promoters contain a single A and B box and are well documented for their role in tRNA and SINE transcription in eukaryotic cells. The genome of Murid herpesvirus 4 (MuHV-4) contains eight polycistronic tRNA-microRNA encoded RNA (TMER) genes that are transcribed from a RNA pol III type 2-like promoter containing triplicated A box elements. Here, we demonstrate that the triplicated A box sequences are required in their entirety to produce functional MuHV-4 miRNAs. We also identify that these RNA pol III type 2-like promoters are conserved in eukaryotic genomes. Human and mouse predicted tRNA genes containing these promoters also show enrichment of alternative RNA pol III transcription termination sequences and are predicted to give rise to longer tRNA primary transcripts.

Developmental roles of Drosophila tRNA processing endonuclease RNase ZL as revealed with a conditional rescue system

Drosophila RNase ZL (dRNaseZ) belongs to a family of endoribonucleases with a major role in tRNA 3′-end processing. The biochemical function of RNase ZL is conserved from yeast to human. Here we present a study of its biological function during Drosophila development. In flies, dRNaseZ provides a non-redundant function, as the RNZED24 knockout (KO) mutation causes early larval lethality. Mosaic and conditional rescue techniques were employed to determine dRNaseZ requirements at later stages. We found that dRNaseZ activity is essential for all phases of fly development that involve cell division, including growth of adult tissue progenitors during larval and metamorphic stages, and gametogenesis in adults. At the cellular level, two major phenotypes were identified—cell growth deficiency in endoreplicating tissues and cell cycle arrest in mitotic tissues. While cell growth and proliferation are both dependant on protein synthesis, the two phenotypes displayed reliance on different dRNaseZ functions. We found that dRNaseZ KO completely blocks tRNA maturation without diminishing the abundance of mature tRNA molecules. Our data indicate that growth arrest of endoreplicating cells is primarily attributed to the relocation of the pool of mature tRNAs into the nuclei causing a decrease in translation efficiency. Mitotically dividing cells appear to be less dependent on translation machinery as they maintain their normal size when deprived of dRNaseZ activity, but rather display a cell cycle arrest at the G2–M transition.

The concept of RNA-assisted protein folding: the role of tRNA.

We suggest that tRNA actively participates in the transfer of 3D information from mRNA to peptides - in addition to its well-known, "classical" role of translating the 3-letter RNA codes into the one letter protein code. The tRNA molecule displays a series of thermodynamically favored configurations during translation, a movement which places the codon and coded amino acids in proximity to each other and make physical contact between some amino acids and their codons possible. This specific codon-amino acid interaction of some selected amino acids is necessary for the transfer of spatial information from mRNA to coded proteins, and is known as RNA-assisted protein folding.

Roles of tRNA in cell wall biosynthesis.

Recent research into various aspects of bacterial metabolism such as cell wall and antibiotic synthesis, degradation pathways, cellular stress, and amino acid biosynthesis has elucidated roles of aminoacyl-transfer ribonucleic acid (aa-tRNA) outside of translation. Although the two enzyme families responsible for cell wall modifications, aminoacyl-phosphatidylglycerol synthases (aaPGSs) and Fem, were discovered some time ago, they have recently become of intense interest for their roles in the antimicrobial resistance of pathogenic microorganisms. The addition of positively charged amino acids to phosphatidylglycerol (PG) by aaPGSs neutralizes the lipid bilayer making the bacteria less susceptible to positively charged antimicrobial agents. Fem transferases utilize aa-tRNA to form peptide bridges that link strands of peptidoglycan. These bridges vary among the bacterial species in which they are present and play a role in resistance to antibiotics that target the cell wall. Additionally, the formation of truncated peptides results in shorter peptide bridges and loss of branched linkages which makes bacteria more susceptible to antimicrobials. A greater understanding of the structure and substrate specificity of this diverse enzymatic family is necessary to aid current efforts in designing potential bactericidal agents. These two enzyme families are linked only by the substrate with which they modify the cell wall, aa-tRNA; their structure, cell wall modification processes and the physiological changes they impart on the bacterium differ greatly.

Transfer RNA-derived Small RNAs: Degradation Fragments or Novel Regulatory Molecules?*

Transfer RNAs (tRNAs) with classic clover structure, have been researched as a key component of cellular protein synthesis machine for decades. However, the pace of progress in the understanding of tRNA function is still staggering, especially in the role of tRNA as the precursor of potential molecules to regulate gene expression globally. Several recent studies suggest that some tRNA-derived small RNAs have been detected in many cell lines by deep sequencing. These cleavage products of tRNAs are indicated to interact with some important protein factors like Dicer and Ago family in microRNA processing system. In additional, luciferase reporter assay results implied that the small RNAs derived from tRNA might play some roles in response to the stress as regulatory molecules with microRNA-like features. If their regulatory functions were to be confirmed by more evidence, these newly revealed RNAs would provide expanding insight into the repertoire of small noncoding RNAs.

The role of tRNA and ribosome competition in coupling the expression of different mRNAs in Saccharomyces cerevisiae

Protein synthesis translates information from messenger RNAs into functional proteomes. Because of the finite nature of the resources required by the translational machinery, both the overall protein synthesis activity of a cell and activity on individual mRNAs are controlled by the allocation of limiting resources. Upon introduction of heterologous sequences into an organism—for example for the purposes of bioprocessing or synthetic biology—limiting resources may also become overstretched, thus negatively affecting both endogenous and heterologous gene expression. In this study, we present a mean-field model of translation in Saccharomyces cerevisiae for the investigation of two particular translational resources, namely ribosomes and aminoacylated tRNAs. We firstly use comparisons of experiments with heterologous sequences and simulations of the same conditions to calibrate our model, and then analyse the behaviour of the translational system in yeast upon introduction of different types of heterologous sequences. Our main findings are that: competition for ribosomes, rather than tRNAs, limits global translation in this organism; that tRNA aminoacylation levels exert, at most, weak control over translational activity; and that decoding speeds and codon adaptation exert strong control over local (mRNA specific) translation rates.

A role for tRNAHisguanylyltransferase (Thg1)-like proteins fromDictyostelium discoideumin mitochondrial 5′-tRNA editing

Genes with sequence similarity to the yeast tRNA(His) guanylyltransferase (Thg1) gene have been identified in all three domains of life, and Thg1 family enzymes are implicated in diverse processes, ranging from tRNA(His) maturation to 5'-end repair of tRNAs. All of these activities take advantage of the ability of Thg1 family enzymes to catalyze 3'-5' nucleotide addition reactions. Although many Thg1-containing organisms have a single Thg1-related gene, certain eukaryotic microbes possess multiple genes with sequence similarity to Thg1. Here we investigate the activities of four Thg1-like proteins (TLPs) encoded by the genome of the slime mold, Dictyostelium discoideum (a member of the eukaryotic supergroup Amoebozoa). We show that one of the four TLPs is a bona fide Thg1 ortholog, a cytoplasmic G(-1) addition enzyme likely to be responsible for tRNA(His) maturation in D. discoideum. Two other D. discoideum TLPs exhibit biochemical activities consistent with a role for these enzymes in mitochondrial 5'-tRNA editing, based on their ability to efficiently repair the 5' ends of mitochondrial tRNA editing substrates. Although 5'-tRNA editing was discovered nearly two decades ago, the identity of the protein(s) that catalyze this activity has remained elusive. This article provides the first identification of any purified protein that appears to play a role in the 5'-tRNA editing reaction. Moreover, the presence of multiple Thg1 family members in D. discoideum suggests that gene duplication and divergence during evolution has resulted in paralogous proteins that use 3'-5' nucleotide addition reactions for diverse biological functions in the same organism.

TRNA 5′-end repair activities of tRNA His guanylyltransferase (Thg1)-like proteins from Bacteria and Archaea

The tRNAHis guanylyltransferase (Thg1) family comprises a set of unique 3′–5′ nucleotide addition enzymes found ubiquitously in Eukaryotes, where they function in the critical G−1 addition reaction required for tRNAHis maturation. However, in most Bacteria and Archaea, G−1 is genomically encoded; thus post-transcriptional addition of G−1 to tRNAHis is not necessarily required. The presence of highly conserved Thg1-like proteins (TLPs) in more than 40 bacteria and archaea therefore suggests unappreciated roles for TLP-catalyzed 3′–5′ nucleotide addition. Here, we report that TLPs from Bacillus thuringiensis (BtTLP) and Methanosarcina acetivorans (MaTLP) display biochemical properties consistent with a prominent role in tRNA 5′-end repair. Unlike yeast Thg1, BtTLP strongly prefers addition of missing N+1 nucleotides to 5′-truncated tRNAs over analogous additions to full-length tRNA (kcat/KM enhanced 5–160-fold). Moreover, unlike for −1 addition, BtTLP-catalyzed additions to truncated tRNAs are not biased toward addition of G, and occur with tRNAs other than tRNAHis. Based on these distinct biochemical properties, we propose that rather than functioning solely in tRNAHis maturation, bacterial and archaeal TLPs are well-suited to participate in tRNA quality control pathways. These data support more widespread roles for 3′–5′ nucleotide addition reactions in biology than previously expected.

Transferring Death: A Role for tRNA in Apoptosis Regulation

Cytochrome c from mitochondria significantly accelerates the apoptotic process. In this issue of Molecular Cell, Mei et al. show that cytochrome c is itself regulated by binding to tRNA, providing an unanticipated level of control of cell fate decisions.

A rationally engineered misacylating aminoacyl-tRNA synthetase

Information transfer from nucleic acid to protein is mediated by aminoacyl-tRNA synthetases, which catalyze the specific pairings of amino acids with transfer RNAs. Despite copious sequence and structural information on the 22 tRNA synthetase families, little is known of the enzyme signatures that specify amino acid selectivities. Here, we show that transplanting a conserved arginine residue from glutamyl-tRNA synthetase (GluRS) to glutaminyl-tRNA synthetase (GlnRS) improves the K(M) of GlnRS for noncognate glutamate. Two crystal structures of this C229R GlnRS mutant reveal that a conserved twin-arginine GluRS amino acid identity signature cannot be incorporated into GlnRS without disrupting surrounding protein structural elements that interact with the tRNA. Consistent with these findings, we show that cumulative replacement of other primary binding site residues in GlnRS, with those of GluRS, only slightly improves the ability of the GlnRS active site to accommodate glutamate. However, introduction of 22 amino acid replacements and one deletion, including substitution of the entire primary binding site and two surface loops adjacent to the region disrupted in C229R, improves the capacity of Escherichia coli GlnRS to synthesize misacylated Glu-tRNA(Gln) by 16,000-fold. This hybrid enzyme recapitulates the function of misacylating GluRS enzymes found in organisms that synthesize Gln-tRNA(Gln) by an alternative pathway. These findings implicate the RNA component of the contemporary GlnRS-tRNA(Gln) complex in mediating amino acid specificity. This role for tRNA may persist as a relic of primordial cells in which the evolution of the genetic code was driven by RNA-catalyzed amino acid-RNA pairing.