Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation.

In bacterial translational initiation, three initiation factors (IFs 1-3) enable the selection of initiator tRNA and the start codon in the P site of the 30S ribosomal subunit. Here, we report 11 single-particle cryo-electron microscopy (cryoEM) reconstructions of the complex of bacterial 30S subunit with initiator tRNA, mRNA, and IFs 1-3, representing different steps along the initiation pathway. IF1 provides key anchoring points for IF2 and IF3, thereby enhancing their activities. IF2 positions a domain in an extended conformation appropriate for capturing the formylmethionyl moiety charged on tRNA. IF3 and tRNA undergo large conformational changes to facilitate the accommodation of the formylmethionyl-tRNA (fMet-tRNA(fMet)) into the P site for start codon recognition.

Serine Catabolism by SHMT2 Is Required for Proper Mitochondrial Translation Initiation and Maintenance of Formylmethionyl-tRNAs.

Upon glucose restriction, eukaryotic cells upregulate oxidative metabolism to maintain homeostasis. Using genetic screens, we find that the mitochondrial serine hydroxymethyltransferase (SHMT2) is required for robust mitochondrial oxygen consumption and low glucose proliferation. SHMT2 catalyzes the first step in mitochondrial one-carbon metabolism, which, particularly in proliferating cells, produces tetrahydrofolate (THF)-conjugated one-carbon units used in cytoplasmic reactions despite the presence of a parallel cytoplasmic pathway. Impairing cytoplasmic one-carbon metabolism or blocking efflux of one-carbon units from mitochondria does not phenocopy SHMT2 loss, indicating that a mitochondrial THF cofactor is responsible for the observed phenotype. The enzyme MTFMT utilizes one such cofactor, 10-formyl THF, producing formylmethionyl-tRNAs, specialized initiator tRNAs necessary for proper translation of mitochondrially encoded proteins. Accordingly, SHMT2 null cells specifically fail to maintain formylmethionyl-tRNA pools and mitochondrially encoded proteins, phenotypes similar to those observed in MTFMT-deficient patients. These findings provide a rationale for maintaining a compartmentalized one-carbon pathway in mitochondria.

Translation initiation with exotic amino acids using EF-P-responsive artificial initiator tRNA.

Translation initiation using noncanonical initiator substrates with poor peptidyl donor activities, such as N-acetyl-l-proline (AcPro), induces the N-terminal drop-off-reinitiation event. Thereby, the initiator tRNA drops-off from the ribosome and the translation reinitiates from the second amino acid to yield a truncated peptide lacking the N-terminal initiator substrate. In order to suppress this event for the synthesis of full-length peptides, here we have devised a chimeric initiator tRNA, referred to as tRNAiniP, whose D-arm comprises a recognition motif for EF-P, an elongation factor that accelerates peptide bond formation. We have shown that the use of tRNAiniP and EF-P enhances the incorporation of not only AcPro but also d-amino, ß-amino and γ-amino acids at the N-terminus. By optimizing the translation conditions, e.g. concentrations of translation factors, codon sequence and Shine-Dalgarno sequence, we could achieve complete suppression of the N-terminal drop-off-reinitiation for the exotic amino acids and enhance the expression level of full-length peptide up to 1000-fold compared with the use of the ordinary translation conditions.

The ribosomal protein S1-dependent standby site in tisB mRNA consists of a single-stranded region and a 5' structure element.

In bacteria, stable RNA structures that sequester ribosome-binding sites (RBS) impair translation initiation, and thus protein output. In some cases, ribosome standby can overcome inhibition by structure: 30S subunits bind sequence-nonspecifically to a single-stranded region and, on breathing of the inhibitory structure, relocate to the RBS for initiation. Standby can occur over long distances, as in the active, +42 tisB mRNA, encoding a toxin. This mRNA is translationally silenced by an antitoxin sRNA, IstR-1, that base pairs to the standby site. In tisB and other cases, a direct interaction between 30S subunits and a standby site has remained elusive. Based on fluorescence anisotropy experiments, ribosome toeprinting results, in vitro translation assays, and cross-linking-immunoprecipitation (CLIP) in vitro, carried out on standby-proficient and standby-deficient tisB mRNAs, we provide a thorough characterization of the tisB standby site. 30S subunits and ribosomal protein S1 alone display high-affinity binding to standby-competent fluorescein-labeled +42 mRNA, but not to mRNAs that lack functional standby sites. Ribosomal protein S1 is essential for standby, as 30∆S1 subunits do not support standby-dependent toeprints and TisB translation in vitro. S1 alone- and 30S-CLIP followed by RNA-seq mapping shows that the functional tisB standby site consists of the expected single-stranded region, but surprisingly, also a 5'-end stem-loop structure. Removal of the latter by 5'-truncations, or disruption of the stem, abolishes 30S binding and standby activity. Based on the CLIP-read mapping, the long-distance standby effect in +42 tisB mRNA (∼100 nt) is tentatively explained by S1-dependent directional unfolding toward the downstream RBS.

tRNA binding, structure, and localization of the human interferon-induced protein IFIT5.

Interferon-induced proteins, including the largely uncharacterized interferon-induced tetratricopeptide repeat (IFIT) protein family, provide defenses against pathogens. Differing from expectations for tetratricopeptide repeat (TPR) proteins and from human IFIT1, IFIT2, and IFIT3, we show that human IFIT5 recognizes cellular RNA instead of protein partners. In vivo and in vitro, IFIT5 bound to endogenous 5'-phosphate-capped RNAs, including transfer RNAs. The crystal structure of IFIT5 revealed a convoluted intramolecular packing of eight TPRs as a fold that we name the TPR eddy. Additional, non-TPR structural elements contribute to an RNA binding cleft. Instead of general cytoplasmic distribution, IFIT5 concentrated in actin-rich protrusions from the apical cell surface colocalized with the RNA-binding retinoic acid-inducible gene-I (RIG-I). These findings establish compartmentalized cellular RNA binding activity as a mechanism for IFIT5 function and reveal the TPR eddy as a scaffold for RNA recognition.

Rapid formylation of the cellular initiator tRNA population makes a crucial contribution to its exclusive participation at the step of initiation.

Initiator tRNAs (i-tRNAs) possess highly conserved three consecutive GC base pairs (GC/GC/GC, 3GC pairs) in their anticodon stems. Additionally, in bacteria and eukaryotic organelles, the amino acid attached to i-tRNA is formylated by Fmt to facilitate its targeting to 30S ribosomes. Mutations in GC/GC/GC to UA/CG/AU in i-tRNACUA/3GC do not affect its formylation. However, the i-tRNACUA/3GC is non-functional in initiation. Here, we characterised an Escherichia coli strain possessing an amber mutation in its fmt gene (fmtam274), which affords initiation with i-tRNACUA/3GC. Replacement of fmt with fmtam274 in the parent strain results in production of truncated Fmt, accumulation of unformylated i-tRNA, and a slow growth phenotype. Introduction of i-tRNACUA/3GC into the fmtam274 strain restores accumulation of formylated i-tRNAs and rescues the growth defect of the strain. We show that i-tRNACUA/3GC causes a low level suppression of am274 in fmtam274. Low levels of cellular Fmt lead to compromised efficiency of formylation of i-tRNAs, which in turn results in distribution of the charged i-tRNAs between IF2 and EF-Tu allowing the plasmid borne i-tRNACUA/3GC to function at both the initiation and elongation steps. We show that a speedy formylation of i-tRNA population is crucial for its preferential binding (and preventing other tRNAs) into the P-site.

MEHMO syndrome mutation EIF2S3-I259M impairs initiator Met-tRNAiMet binding to eukaryotic translation initiation factor eIF2.

The heterotrimeric eukaryotic translation initiation factor (eIF) 2 plays critical roles in delivering initiator Met-tRNAiMet to the 40S ribosomal subunit and in selecting the translation initiation site. Genetic analyses of patients with MEHMO syndrome, an X-linked intellectual disability syndrome, have identified several unique mutations in the EIF2S3 gene that encodes the γ subunit of eIF2. To gain insights into the molecular consequences of MEHMO syndrome mutations on eIF2 function, we generated a yeast model of the human eIF2γ-I259M mutant, previously identified in a patient with MEHMO syndrome. The corresponding eIF2γ-I318M mutation impaired yeast cell growth and derepressed GCN4 expression, an indicator of defective eIF2-GTP-Met-tRNAiMet complex formation, and, likewise, overexpression of human eIF2γ-I259M derepressed ATF4 messenger RNA translation in human cells. The yeast eIF2γ-I318M mutation also increased initiation from near-cognate start codons. Biochemical analyses revealed a defect in Met-tRNAiMet binding to the mutant yeast eIF2 complexes in vivo and in vitro. Overexpression of tRNAiMet restored Met-tRNAiMet binding to eIF2 in vivo and rescued the growth defect in the eIF2γ-I318M strain. Based on these findings and the structure of eIF2, we propose that the I259M mutation impairs Met-tRNAiMet binding, causing altered control of protein synthesis that underlies MEHMO syndrome.

eIF1 Loop 2 interactions with Met-tRNAi control the accuracy of start codon selection by the scanning preinitiation complex.

The eukaryotic 43S preinitiation complex (PIC), bearing initiator methionyl transfer RNA (Met-tRNAi) in a ternary complex (TC) with eukaryotic initiation factor 2 (eIF2)-GTP, scans the mRNA leader for an AUG codon in favorable context. AUG recognition evokes rearrangement from an open PIC conformation with TC in a "POUT" state to a closed conformation with TC more tightly bound in a "PIN" state. eIF1 binds to the 40S subunit and exerts a dual role of enhancing TC binding to the open PIC conformation while antagonizing the PIN state, necessitating eIF1 dissociation for start codon selection. Structures of reconstituted PICs reveal juxtaposition of eIF1 Loop 2 with the Met-tRNAi D loop in the PIN state and predict a distortion of Loop 2 from its conformation in the open complex to avoid a clash with Met-tRNAi We show that Ala substitutions in Loop 2 increase initiation at both near-cognate UUG codons and AUG codons in poor context. Consistently, the D71A-M74A double substitution stabilizes TC binding to 48S PICs reconstituted with mRNA harboring a UUG start codon, without affecting eIF1 affinity for 40S subunits. Relatively stronger effects were conferred by arginine substitutions; and no Loop 2 substitutions perturbed the rate of TC loading on scanning 40S subunits in vivo. Thus, Loop 2-D loop interactions specifically impede Met-tRNAi accommodation in the PIN state without influencing the POUT mode of TC binding; and Arg substitutions convert the Loop 2-tRNAi clash to an electrostatic attraction that stabilizes PIN and enhances selection of poor start codons in vivo.

The structure of an E. coli tRNAfMet A1-U72 variant shows an unusual conformation of the A1-U72 base pair.

Translation initiation in eukaryotes and archaea involves a methionylated initiator tRNA delivered to the ribosome in a ternary complex with e/aIF2 and GTP. Eukaryotic and archaeal initiator tRNAs contain a highly conserved A1-U72 base pair at the top of the acceptor stem. The importance of this base pair to discriminate initiator tRNAs from elongator tRNAs has been established previously using genetics and biochemistry. However, no structural data illustrating how the A1-U72 base pair participates in the accurate selection of the initiator tRNAs by the translation initiation systems are available. Here, we describe the crystal structure of a mutant E. coli initiator tRNAfMetA1-U72, aminoacylated with methionine, in which the C1:A72 mismatch at the end of the tRNA acceptor stem has been changed to an A1-U72 base pair. Sequence alignments show that the mutant E. coli tRNA is a good mimic of archaeal initiator tRNAs. The crystal structure, determined at 2.8 Å resolution, shows that the A1-U72 pair adopts an unusual arrangement. A1 is in a syn conformation and forms a single H-bond interaction with U72 This interaction requires protonation of the N1 atom of A1 Moreover, the 5' phosphoryl group folds back into the major groove of the acceptor stem and interacts with the N7 atom of G2 A possible role of this unusual geometry of the A1-U72 pair in the recognition of the initiator tRNA by its partners during eukaryotic and archaeal translation initiation is discussed.

The contribution of the C5 protein subunit of Escherichia coli ribonuclease P to specificity for precursor tRNA is modulated by proximal 5' leader sequences.

Recognition of RNA by RNA processing enzymes and RNA binding proteins often involves cooperation between multiple subunits. However, the interdependent contributions of RNA and protein subunits to molecular recognition by ribonucleoproteins are relatively unexplored. RNase P is an endonuclease that removes 5' leaders from precursor tRNAs and functions in bacteria as a dimer formed by a catalytic RNA subunit (P RNA) and a protein subunit (C5 in E. coli). The P RNA subunit contacts the tRNA body and proximal 5' leader sequences [N(-1) and N(-2)] while C5 binds distal 5' leader sequences [N(-3) to N(-6)]. To determine whether the contacts formed by P RNA and C5 contribute independently to specificity or exhibit cooperativity or anti-cooperativity, we compared the relative kcat/Km values for all possible combinations of the six proximal 5' leader nucleotides (n = 4096) for processing by the E. coli P RNA subunit alone and by the RNase P holoenzyme. We observed that while the P RNA subunit shows specificity for 5' leader nucleotides N(-2) and N(-1), the presence of the C5 protein reduces the contribution of P RNA to specificity, but changes specificity at N(-2) and N(-3). The results reveal that the contribution of C5 protein to RNase P processing is controlled by the identity of N(-2) in the pre-tRNA 5' leader. The data also clearly show that pairing of the 5' leader with the 3' ACCA of tRNA acts as an anti-determinant for RNase P cleavage. Comparative analysis of genomically encoded E. coli tRNAs reveals that both anti-determinants are subject to negative selection in vivo.

The initiation of mammalian protein synthesis and mRNA scanning mechanism.

During translation initiation in eukaryotes, the small ribosomal subunit binds messenger RNA at the 5' end and scans in the 5' to 3' direction to locate the initiation codon, form the 80S initiation complex and start protein synthesis. This simple, yet intricate, process is guided by multiple initiation factors. Here we determine the structures of three complexes of the small ribosomal subunit that represent distinct steps in mammalian translation initiation. These structures reveal the locations of eIF1, eIF1A, mRNA and initiator transfer RNA bound to the small ribosomal subunit and provide insights into the details of translation initiation specific to eukaryotes. Conformational changes associated with the captured functional states reveal the dynamics of the interactions in the P site of the ribosome. These results have functional implications for the mechanism of mRNA scanning.

Hidden specificity in an apparently nonspecific RNA-binding protein.

Nucleic-acid-binding proteins are generally viewed as either specific or nonspecific, depending on characteristics of their binding sites in DNA or RNA. Most studies have focused on specific proteins, which identify cognate sites by binding with highest affinities to regions with defined signatures in sequence, structure or both. Proteins that bind to sites devoid of defined sequence or structure signatures are considered nonspecific. Substrate binding by these proteins is poorly understood, and it is not known to what extent seemingly nonspecific proteins discriminate between different binding sites, aside from those sequestered by nucleic acid structures. Here we systematically examine substrate binding by the apparently nonspecific RNA-binding protein C5, and find clear discrimination between different binding site variants. C5 is the protein subunit of the transfer RNA processing ribonucleoprotein enzyme RNase P from Escherichia coli. The protein binds 5' leaders of precursor tRNAs at a site without sequence or structure signatures. We measure functional binding of C5 to all possible sequence variants in its substrate binding site, using a high-throughput sequencing kinetics approach (HITS-KIN) that simultaneously follows processing of thousands of RNA species. C5 binds different substrate variants with affinities varying by orders of magnitude. The distribution of functional affinities of C5 for all substrate variants resembles affinity distributions of highly specific nucleic acid binding proteins. Unlike these specific proteins, C5 does not bind its physiological RNA targets with the highest affinity, but with affinities near the median of the distribution, a region that is not associated with a sequence signature. We delineate defined rules governing substrate recognition by C5, which reveal specificity that is hidden in cellular substrates for RNase P. Our findings suggest that apparently nonspecific and specific RNA-binding modes may not differ fundamentally, but represent distinct parts of common affinity distributions.

Two highly conserved features of bacterial initiator tRNAs license them to pass through distinct checkpoints in translation initiation.

Eubacterial translation initiation involves assembly of tRNAfMet, mRNA, initiation factors (IFs) and 30S ribosome in a 30S pre-initiation complex (30S pre-IC), which rearranges and joins 50S ribosome to form 70S IC. Upon releasing IFs, 70S IC becomes elongation-competent 70S. The direct recruitment of initiator tRNA (tRNAfMet) into the ribosomal P-site, crucial in accurate initiation of translation, is attributed to two conserved features of tRNAfMet: (i) formylation of amino acid attached to it and, (ii) the presence of three consecutive G-C base pairs (3GC base pairs) in the anticodon stem. However, the precise roles of these two conserved features of tRNAfMet during the various steps of initiation remain unclear. Using natural and engineered tRNAs, we show that the 3GC pairs license tRNAfMet transitions from 30S to 70S IC and then to elongation-competent 70S by release of IF3. Of the 3GC pairs, the middle GC pair (G30-C40), or merely G30 (in a specific context) suffices in this role and is essential for the sustenance of Escherichia coli. Furthermore, rescue of formylase deficient E. coli by overproduced tRNAfMet reveals that the feature of formylation licenses initial targeting of tRNAfMet to 30S ribosome

Structure of a 30S pre-initiation complex stalled by GE81112 reveals structural parallels in bacterial and eukaryotic protein synthesis initiation pathways.

In bacteria, the start site and the reading frame of the messenger RNA are selected by the small ribosomal subunit (30S) when the start codon, typically an AUG, is decoded in the P-site by the initiator tRNA in a process guided and controlled by three initiation factors. This process can be efficiently inhibited by GE81112, a natural tetrapeptide antibiotic that is highly specific toward bacteria. Here GE81112 was used to stabilize the 30S pre-initiation complex and obtain its structure by cryo-electron microscopy. The results obtained reveal the occurrence of changes in both the ribosome conformation and initiator tRNA position that may play a critical role in controlling translational fidelity. Furthermore, the structure highlights similarities with the early steps of initiation in eukaryotes suggesting that shared structural features guide initiation in all kingdoms of life.

NSUN3 methylase initiates 5-formylcytidine biogenesis in human mitochondrial tRNA(Met).

In human mitochondria, the AUA codon encodes methionine via a mitochondrial transfer RNA for methionine (mt-tRNA(Met)) that contains 5-formylcytidine (f(5)C) at the first position of the anticodon (position 34). f(5)C34 is required for deciphering the AUA codon during protein synthesis. Until now, the biogenesis and physiological role of f(5)C34 were unknown. We demonstrate that biogenesis of f(5)C34 is initiated by S-adenosylmethionine (AdoMet)-dependent methylation catalyzed by NSUN3, a putative methyltransferase in mitochondria. NSUN3-knockout cells showed strong reduction in mitochondrial protein synthesis and reduced oxygen consumption, leading to deficient mitochondrial activity. We reconstituted formation of 5-methylcytidine (m(5)C) at position 34 (m(5)C34) on mt-tRNA(Met) with recombinant NSUN3 in the presence of AdoMet, demonstrating that NSUN3-mediated m(5)C34 formation initiates f(5)C34 biogenesis. We also found two disease-associated point mutations in mt-tRNA(Met) that impaired m(5)C34 formation by NSUN3, indicating that a lack of f(5)C34 has pathological consequences.

Messing up translation from the start: How AtaT inhibits translation initiation in E. coli.

Toxin-antitoxin systems (TA) are widespread in bacteria and archea. They are commonly found in chromosomes and mobile genetic elements. These systems move from different genomic locations and bacterial hosts through horizontal gene transfer, using mobile elements as vehicles. Their potential roles in bacterial physiology are still a matter of debate in the field. The mechanisms of action of different toxin families have been deciphered at the molecular level. Intriguingly, the vast majority of these toxins target protein synthesis. They use a variety of molecular mechanisms and inhibit nearly every step of the translation process. Recently, we have identified a novel toxin, AtaT, presenting acetyltransferase activity. 1 Our work uncovered the molecular activity of AtaT: it specifically acetylates the methionine moiety on the initiator Met-tRNAfMet. This modification drastically impairs recognition by initiation factor 2 (IF2), thereby inhibiting the initiation step of translation.

IF2 and unique features of initiator tRNAfMet help establish the translational reading frame.

Translation begins at AUG, GUG, or UUG codons in bacteria. Start codon recognition occurs in the P site, which may help explain this first-position degeneracy. However, the molecular basis of start codon specificity remains unclear. In this study, we measured the codon dependence of 30S•mRNA•tRNAfMet and 30S•mRNA•tRNAMet complex formation. We found that complex stability varies over a large range with initiator tRNAfMet, following the same trend as reported previously for initiation rate in vivo (AUG > GUG, UUG > CUG, AUC, AUA > ACG). With elongator tRNAMet, the codon dependence of binding differs qualitatively, with virtually no discrimination between GUG and CUG. A unique feature of initiator tRNAfMet is a series of three G-C basepairs in the anticodon stem, which are known to be important for efficient initiation in vivo. A mutation targeting the central of these G-C basepairs causes the mRNA binding specificity pattern to change in a way reminiscent of elongator tRNAMet. Unexpectedly, for certain complexes containing fMet-tRNAfMet, we observed mispositioning of mRNA, such that codon 2 is no longer programmed in the A site. This mRNA mispositioning is exacerbated by the anticodon stem mutation and suppressed by IF2. These findings suggest that both IF2 and the unique anticodon stem of fMet-tRNAfMet help constrain mRNA positioning to set the correct reading frame during initiation.

VapCs of Mycobacterium tuberculosis cleave RNAs essential for translation.

The major human pathogen Mycobacterium tuberculosis can survive in the host organism for decades without causing symptoms. A large cohort of Toxin-Antitoxin (TA) modules contribute to this persistence. Of these, 48 TA modules belong to the vapBC (virulence associated protein) gene family. VapC toxins are PIN domain endonucleases that, in enterobacteria, inhibit translation by site-specific cleavage of initiator tRNA. In contrast, VapC20 of M. tuberculosis inhibits translation by site-specific cleavage of the universally conserved Sarcin-Ricin loop (SRL) in 23S rRNA. Here we identify the cellular targets of 12 VapCs from M. tuberculosis by applying UV-crosslinking and deep sequencing. Remarkably, these VapCs are all endoribonucleases that cleave RNAs essential for decoding at the ribosomal A-site. Eleven VapCs cleave specific tRNAs while one exhibits SRL cleavage activity. These findings suggest that multiple vapBC modules contribute to the survival of M. tuberculosis in its human host by reducing the level of translation.

Structural and functional insights into tRNA binding and adenosine N1-methylation by an archaeal Trm10 homologue.

Purine nucleosides on position 9 of eukaryal and archaeal tRNAs are frequently modified in vivo by the post-transcriptional addition of a methyl group on their N1 atom. The methyltransferase Trm10 is responsible for this modification in both these domains of life. While certain Trm10 orthologues specifically methylate either guanosine or adenosine at position 9 of tRNA, others have a dual specificity. Until now structural information about this enzyme family was only available for the catalytic SPOUT domain of Trm10 proteins that show specificity toward guanosine. Here, we present the first crystal structure of a full length Trm10 orthologue specific for adenosine, revealing next to the catalytic SPOUT domain also N- and C-terminal domains. This structure hence provides crucial insights in the tRNA binding mechanism of this unique monomeric family of SPOUT methyltransferases. Moreover, structural comparison of this adenosine-specific Trm10 orthologue with guanosine-specific Trm10 orthologues suggests that the N1 methylation of adenosine relies on additional catalytic residues.

Initiator tRNA genes template the 3' CCA end at high frequencies in bacteria.

BACKGROUND: While the CCA sequence at the mature 3' end of tRNAs is conserved and critical for translational function, a genetic template for this sequence is not always contained in tRNA genes. In eukaryotes and Archaea, the CCA ends of tRNAs are synthesized post-transcriptionally by CCA-adding enzymes. In Bacteria, tRNA genes template CCA sporadically. RESULTS: In order to understand the variation in how prokaryotic tRNA genes template CCA, we re-annotated tRNA genes in tRNAdb-CE database version 0.8. Among 132,129 prokaryotic tRNA genes, initiator tRNA genes template CCA at the highest average frequency (74.1%) over all functional classes except selenocysteine and pyrrolysine tRNA genes (88.1% and 100% respectively). Across bacterial phyla and a wide range of genome sizes, many lineages exist in which predominantly initiator tRNA genes template CCA. Convergent and parallel retention of CCA templating in initiator tRNA genes evolved in independent histories of reductive genome evolution in Bacteria. Also, in a majority of cyanobacterial and actinobacterial genera, predominantly initiator tRNA genes template CCA. We also found that a surprising fraction of archaeal tRNA genes template CCA. CONCLUSIONS: We suggest that cotranscriptional synthesis of initiator tRNA CCA 3' ends can complement inefficient processing of initiator tRNA precursors, "bootstrap" rapid initiation of protein synthesis from a non-growing state, or contribute to an increase in cellular growth rates by reducing overheads of mass and energy to maintain nonfunctional tRNA precursor pools. More generally, CCA templating in structurally non-conforming tRNA genes can afford cells robustness and greater plasticity to respond rapidly to environmental changes and stimuli.