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.

Development of Assay Systems for Amber Codon Decoding at the Steps of Initiation and Elongation in Mycobacteria.

Genetic analysis of the mechanism of protein synthesis in Gram-positive bacteria has remained largely unexplored because of the unavailability of appropriate in vivo assay systems. We developed chloramphenicol acetyltransferase (CAT)-based in vivo reporter systems to study translation initiation and elongation in Mycobacterium smegmatis The CAT reporters utilize specific decoding of amber codons by mutant initiator tRNA (i-tRNA, metU) molecules containing a CUA anticodon (metUCUA). The assay systems allow structure-function analyses of tRNAs without interfering with the cellular protein synthesis and function with or without the expression of heterologous GlnRS from Escherichia coli We show that despite their naturally occurring slow-growth phenotypes, the step of i-tRNA formylation is vital in translation initiation in mycobacteria and that formylation-deficient i-tRNA mutants (metUCUA/A1, metUCUA/G72, and metUCUA/G72G73) with a Watson-Crick base pair at the 1·72 position participate in elongation. In the absence of heterologous GlnRS expression, the mutant tRNAs are predominantly aminoacylated (glutamylated) by nondiscriminating GluRS. Acid urea gels show complete transamidation of the glutamylated metUCUA/G72G73 tRNA to its glutaminylated form (by GatCAB) in M. smegmatis In contrast, the glutamylated metUCUA/G72 tRNA did not show a detectable level of transamidation. Interestingly, the metUCUA/A1 mutant showed an intermediate activity of transamidation and accumulated in both glutamylated and glutaminylated forms. These observations suggest important roles for the discriminator base position and/or a weak Watson-Crick base pair at 1·72 for in vivo recognition of the glutamylated tRNAs by M. smegmatis GatCAB.IMPORTANCE Genetic analysis of the translational apparatus in Gram-positive bacteria has remained largely unexplored because of the unavailability of appropriate in vivo assay systems. We developed chloramphenicol acetyltransferase (CAT)-based reporters which utilize specific decoding of amber codons by mutant tRNAs at the steps of initiation and/or elongation to allow structure-function analysis of the translational machinery. We show that formylation of the initiator tRNA (i-tRNA) is crucial even for slow-growing bacteria and that i-tRNA mutants with a CUA anticodon are aminoacylated by nondiscriminating GluRS. The discriminator base position, and/or a weak Watson-Crick base pair at the top of the acceptor stem, provides important determinants for transamidation of the i-tRNA-attached Glu to Gln by the mycobacterial GatCAB.

Structure and dynamics study of translation initiation factor 1 from Staphylococcus aureus suggests its RNA binding mode.

Translation initiation, the rate-limiting step in the protein synthesis, is tightly regulated. As one of the translation initiation factors, translation initiation factor 1 (IF1) plays crucial roles not only in translation but also in many cellular processes that are important for genomic stability, such as the activity of RNA chaperones. Here, we characterize the RNA interactions and dynamics of IF1 from Staphylococcus aureus Mu50 (IF1Sa) by NMR spectroscopy. First, the NMR-derived solution structure of IF1Sa revealed that IF1Sa adopts an oligonucleotide/oligosaccharide binding (OB)-fold. Structural comparisons showed large deviations in the α-helix and the following loop, which are potential RNA-binding regions of the OB-fold, as well as differences in the electrostatic potential surface among bacterial IF1s. Second, the 15N NMR relaxation data for IF1Sa indicated the flexible nature of the α-helix and the following loop region of IF1Sa. Third, RNA-binding properties were studied using FP assays and NMR titrations. FP binding assays revealed that IF1Sa binds to RNAs with moderate affinity. In combination with the structural analysis, the NMR titration results revealed the RNA binding sites. Taken together, these results show that IF1Sa binds RNAs with moderate binding affinity via the residues that occupy the large surface area of its ß-barrel. These findings suggest that IF1Sa is likely to bind RNA in various conformations rather than only at a specific site and indicate that the flexible RNA binding mode of IF1Sa is necessary for its interaction with various RNA substrates.

Precise and reliable gene expression via standard transcription and translation initiation elements.

An inability to reliably predict quantitative behaviors for novel combinations of genetic elements limits the rational engineering of biological systems. We developed an expression cassette architecture for genetic elements controlling transcription and translation initiation in Escherichia coli: transcription elements encode a common mRNA start, and translation elements use an overlapping genetic motif found in many natural systems. We engineered libraries of constitutive and repressor-regulated promoters along with translation initiation elements following these definitions. We measured activity distributions for each library and selected elements that collectively resulted in expression across a 1,000-fold observed dynamic range. We studied all combinations of curated elements, demonstrating that arbitrary genes are reliably expressed to within twofold relative target expression windows with ∼93% reliability. We expect the genetic element definitions validated here can be collectively expanded to create collections of public-domain standard biological parts that support reliable forward engineering of gene expression at genome scales.

Quantitative estimation of activity and quality for collections of functional genetic elements.

The practice of engineering biology now depends on the ad hoc reuse of genetic elements whose precise activities vary across changing contexts. Methods are lacking for researchers to affordably coordinate the quantification and analysis of part performance across varied environments, as needed to identify, evaluate and improve problematic part types. We developed an easy-to-use analysis of variance (ANOVA) framework for quantifying the performance of genetic elements. For proof of concept, we assembled and analyzed combinations of prokaryotic transcription and translation initiation elements in Escherichia coli. We determined how estimation of part activity relates to the number of unique element combinations tested, and we show how to estimate expected ensemble-wide part activity from just one or two measurements. We propose a new statistic, biomolecular part 'quality', for tracking quantitative variation in part performance across changing contexts.

Back to translation: removal of aIF2 from the 5'-end of mRNAs by translation recovery factor in the crenarchaeon Sulfolobus solfataricus.

The translation initiation factor aIF2 of the crenarchaeon Sulfolobus solfataricus (Sso) recruits initiator tRNA to the ribosome and stabilizes mRNAs by binding via the γ-subunit to their 5'-triphosphate end. It has been hypothesized that the latter occurs predominantly during unfavorable growth conditions, and that aIF2 or aIF2-γ is released on relief of nutrient stress to enable in particular anew translation of leaderless mRNAs. As leaderless mRNAs are prevalent in Sso and aIF2-γ bound to the 5'-end of a leaderless RNA inhibited ribosome binding in vitro, we aimed at elucidating the mechanism underlying aIF2/aIF2-γ recycling from mRNAs. We have identified a protein termed Trf (translation recovery factor) that co-purified with trimeric aIF2 during outgrowth of cells from prolonged stationary phase. Subsequent in vitro studies revealed that Trf triggers the release of trimeric aIF2 from RNA, and that Trf directly interacts with the aIF2-γ subunit. The importance of Trf is further underscored by an impaired protein synthesis during outgrowth from stationary phase in a Sso trf deletion mutant.

The conserved GTPase LepA contributes mainly to translation initiation in Escherichia coli.

LepA is a paralog of EF-G found in all bacteria. Deletion of lepA confers no obvious growth defect in Escherichia coli, and the physiological role of LepA remains unknown. Here, we identify nine strains (ΔdksA, ΔmolR1, ΔrsgA, ΔtatB, ΔtonB, ΔtolR, ΔubiF, ΔubiG or ΔubiH) in which ΔlepA confers a synthetic growth phenotype. These strains are compromised for gene regulation, ribosome assembly, transport and/or respiration, indicating that LepA contributes to these functions in some way. We also use ribosome profiling to deduce the effects of LepA on translation. We find that loss of LepA alters the average ribosome density (ARD) for hundreds of mRNA coding regions in the cell, substantially reducing ARD in many cases. By contrast, only subtle and codon-specific changes in ribosome distribution along mRNA are seen. These data suggest that LepA contributes mainly to the initiation phase of translation. Consistent with this interpretation, the effect of LepA on ARD is related to the sequence of the Shine-Dalgarno region. Global perturbation of gene expression in the ΔlepA mutant likely explains most of its phenotypes.

Bacteriophage T7 protein kinase: Site of inhibitory autophosphorylation, and use of dephosphorylated enzyme for efficient modification of protein in vitro.

Bacteriophage T7 encodes a serine/threonine-specific protein kinase that phosphorylates multiple cellular proteins during infection of Escherichia coli. Recombinant T7 protein kinase (T7PK), normally purified in phosphorylated form, exhibits a modest level of phosphotransferase activity. A procedure is described that provides dephosphorylated T7PK with an enhanced ability to phosphorylate protein substrates, including translation initiation factor IF1 and the nuclease domain of ribonuclease III. Mass spectrometric analysis identified Thr12 as the site of IF1 phosphorylation in vitro. T7PK undergoes Mg(2+)-dependent autophosphorylation on Ser216 in vitro, which also is modified in vivo. The inability to isolate the presumptive autophosphorylation-resistant T7PK Ser216Ala mutant indicates a toxicity of the phosphotransferase activity and suggests a role for Ser216 modification in limiting T7PK activity during infection.

Genotyping-by-sequencing-based identification of Arabidopsis pattern recognition receptor RLP32 recognizing proteobacterial translation initiation factor IF1.

Activation of plant pattern-triggered immunity (PTI) relies on the recognition of microbe-derived structures, termed patterns, through plant-encoded surface-resident pattern recognition receptors (PRRs). We show that proteobacterial translation initiation factor 1 (IF1) triggers PTI in Arabidopsis thaliana and related Brassicaceae species. Unlike for most other immunogenic patterns, IF1 elicitor activity cannot be assigned to a small peptide epitope, suggesting that tertiary fold features are required for IF1 receptor activation. We have deployed natural variation in IF1 sensitivity to identify Arabidopsis leucine-rich repeat (LRR) receptor-like protein 32 (RLP32) as IF1 receptor using a restriction site-associated DNA sequencing approach. RLP32 confers IF1 sensitivity to rlp32 mutants, IF1-insensitive Arabidopsis accessions and IF1-insensitive Nicotiana benthamiana, binds IF1 specifically and forms complexes with LRR receptor kinases SOBIR1 and BAK1 to mediate signaling. Similar to other PRRs, RLP32 confers resistance to Pseudomonas syringae, highlighting an unexpectedly complex array of bacterial pattern sensors within a single plant species.

Role of Escherichia coli YbeY, a highly conserved protein, in rRNA processing.

The UPF0054 protein family is highly conserved with homologues present in nearly every sequenced bacterium. In some bacteria, the respective gene is essential, while in others its loss results in a highly pleiotropic phenotype. Despite detailed structural studies, a cellular role for this protein family has remained unknown. We report here that deletion of the Escherichia coli homologue, YbeY, causes striking defects that affect ribosome activity, translational fidelity and ribosome assembly. Mapping of 16S, 23S and 5S rRNA termini reveals that YbeY influences the maturation of all three rRNAs, with a particularly strong effect on maturation at both the 5'- and 3'-ends of 16S rRNA as well as maturation of the 5'-termini of 23S and 5S rRNAs. Furthermore, we demonstrate strong genetic interactions between ybeY and rnc (encoding RNase III), ybeY and rnr (encoding RNase R), and ybeY and pnp (encoding PNPase), further suggesting a role for YbeY in rRNA maturation. Mutation of highly conserved amino acids in YbeY, allowed the identification of two residues (H114, R59) that were found to have a significant effect in vivo. We discuss the implications of these findings for rRNA maturation and ribosome assembly in bacteria.

Function and ribosomal localization of aIF6, a translational regulator shared by archaea and eukarya.

The translation factor IF6 is shared by the Archaea and the Eukarya, but is not found in Bacteria. The properties of eukaryal IF6 (eIF6) have been extensively studied, but remain somewhat elusive. eIF6 behaves as a ribosome-anti-association factor and is involved in miRNA-mediated gene silencing; however, it also seems to participate in ribosome synthesis and export. Here we have determined the function and ribosomal localization of the archaeal (Sulfolobus solfataricus) IF6 homologue (aIF6). We find that aIF6 binds specifically to the 50S ribosomal subunits, hindering the formation of 70S ribosomes and strongly inhibiting translation. aIF6 is uniformly expressed along the cell cycle, but it is upregulated following both cold- and heat shock. The aIF6 ribosomal binding site lies in the middle of the 30-S interacting surface of the 50S subunit, including a number of critical RNA and protein determinants involved in subunit association. The data suggest that the IF6 protein evolved in the archaeal-eukaryal lineage to modulate translational efficiency under unfavourable environmental conditions, perhaps acquiring additional functions during eukaryotic evolution.

Crystal structure of TTHA0061, an uncharacterized protein from Thermus thermophilus HB8, reveals a novel fold.

The crystal structure of an uncharacterized protein TTHA0061 from Thermus thermophilus HB8, was determined and refined to 1.8 A by a single wavelength anomalous dispersion (SAD) method. The structural analysis and comparison of TTHA0061 with other existing structures in the Protein Data Bank (PDB) revealed a novel fold, suggesting that this protein may belong to a translation initiation factor or ribosomal protein family. Differential scanning calorimetry analysis suggested that the thermostability of TTHA0061 increased at pH ranges of 5.8-6.2, perhaps due to the abundance of glutamic acid residues.

Crystal structure of the translation recovery factor Trf from Sulfolobus solfataricus.

During translation initiation, the heterotrimeric archaeal translation initiation factor 2 (aIF2) recruits the initiator tRNAi to the small ribosomal subunit. In the stationary growth phase and/or during nutrient stress, Sulfolobus solfataricus aIF2 has a second function: It protects leaderless mRNAs against degradation by binding to their 5'-ends. The S. solfataricus protein Sso2509 is a translation recovery factor (Trf) that interacts with aIF2 and is responsible for the release of aIF2 from bound mRNAs, thereby enabling translation re-initiation. It is a member of the domain of unknown function 35 (DUF35) protein family and is conserved in Sulfolobales as well as in other archaea. Here, we present the X-ray structure of S. solfataricus Trf solved to a resolution of 1.65 Å. Trf is composed of an N-terminal rubredoxin-like domain containing a bound zinc ion and a C-terminal oligosaccharide/oligonucleotide binding fold domain. The Trf structure reveals putative mRNA binding sites in both domains. Surprisingly, the Trf protein is structurally but not sequentially very similar to proteins linked to acyl-CoA utilization-for example, the Sso2064 protein from S. solfataricus-as well as to scaffold proteins found in the acetoacetyl-CoA thiolase/high-mobility group-CoA synthase complex of the archaeon Methanothermococcus thermolithotrophicus and in a steroid side-chain-cleaving aldolase complex from the bacterium Thermomonospora curvata. This suggests that members of the DUF35 protein family are able to act as scaffolding and binding proteins in a wide variety of biological processes.

Translation Inhibition by Rocaglates Activates a Species-Specific Cell Death Program in the Emerging Fungal Pathogen Candida auris.

Fungal infections are a major contributor to infectious disease-related deaths worldwide. Recently, global emergence of the fungal pathogen Candida auris has caused considerable concern because most C. auris isolates are resistant to fluconazole, the most commonly administered antifungal, and some isolates are resistant to drugs from all three major antifungal classes. To identify novel agents with bioactivity against C. auris, we screened 2,454 compounds from a diversity-oriented synthesis collection. Of the five hits identified, most shared a common rocaglate core structure and displayed fungicidal activity against C. auris These rocaglate hits inhibited translation in C. auris but not in its pathogenic relative Candida albicans Species specificity was contingent on variation at a single amino acid residue in Tif1, a fungal member of the eukaryotic initiation factor 4A (eIF4A) family of translation initiation factors known to be targeted by rocaglates. Rocaglate-mediated inhibition of translation in C. auris activated a cell death program characterized by loss of mitochondrial membrane potential, increased caspase-like activity, and disrupted vacuolar homeostasis. In a rocaglate-sensitized C. albicans mutant engineered to express translation initiation factor 1 (Tif1) with the variant amino acid that we had identified in C. auris, translation was inhibited but no programmed cell death phenotypes were observed. This surprising finding suggests divergence between these related fungal pathogens in their pathways of cellular responses to translation inhibition. From a therapeutic perspective, the chemical biology that we have uncovered reveals species-specific vulnerability in C. auris and identifies a promising target for development of new, mechanistically distinct antifungals in the battle against this emerging pathogen.IMPORTANCE Emergence of the fungal pathogen Candida auris has ignited intrigue and alarm within the medical community and the public at large. This pathogen is unusually resistant to antifungals, threatening to overwhelm current management options. By screening a library of structurally diverse molecules, we found that C. auris is surprisingly sensitive to translation inhibition by a class of compounds known as rocaglates (also known as flavaglines). Despite the high level of conservation across fungi in their protein synthesis machinery, these compounds inhibited translation initiation and activated a cell death program in C. auris but not in its relative Candida albicans Our findings highlight a surprising divergence across the cell death programs operating in Candida species and underscore the need to understand the specific biology of a pathogen in attempting to develop more-effective treatments against it.

Unique Shine-Dalgarno Sequences in Cyanobacteria and Chloroplasts Reveal Evolutionary Differences in Their Translation Initiation.

Microorganisms require efficient translation to grow and replicate rapidly, and translation is often rate-limited by initiation. A prominent feature that facilitates translation initiation in bacteria is the Shine-Dalgarno (SD) sequence. However, there is much debate over its conservation in Cyanobacteria and in chloroplasts which presumably originated from endosymbiosis of ancient Cyanobacteria. Elucidating the utilization of SD sequences in Cyanobacteria and in chloroplasts is therefore important to understand whether 1) SD role in Cyanobacterial translation has been reduced prior to chloroplast endosymbiosis or 2) translation in Cyanobacteria and in plastid has been subjected to different evolutionary pressures. To test these alternatives, we employed genomic, proteomic, and transcriptomic data to trace differences in SD usage among Synechocystis species, Microcystis aeruginosa, cyanophages, Nicotiana tabacum chloroplast, and Arabidopsis thaliana chloroplast. We corrected their mis-annotated 16S rRNA 3' terminus using an RNA-Seq-based approach to determine their SD/anti-SD locational constraints using an improved measurement DtoStart. We found that cyanophages well-mimic Cyanobacteria in SD usage because both have been under the same selection pressure for SD-mediated initiation. Whereas chloroplasts lost this similarity because the need for SD-facilitated initiation has been reduced in plastids having much reduced genome size and different ribosomal proteins as a result of host-symbiont coevolution. Consequently, SD sequence significantly increases protein expression in Cyanobacteria but not in chloroplasts, and only Cyanobacterial genes compensate for a lack of SD sequence by having weaker secondary structures at the 5' UTR. Our results suggest different evolutionary pressures operate on translation initiation in Cyanobacteria and in chloroplast.

Staphylococcus aureus responds to allicin by global S-thioallylation - Role of the Brx/BSH/YpdA pathway and the disulfide reductase MerA to overcome allicin stress.

The prevalence of methicillin-resitant Staphylococcus aureus (MRSA) in hospitals and the community poses an increasing health burden, which requires the discovery of alternative antimicrobials. Allicin (diallyl thiosulfinate) from garlic exhibits broad-spectrum antimicrobial activity against many multidrug resistant bacteria. The thiol-reactive mode of action of allicin involves its S-thioallylations of low molecular weight (LMW) thiols and protein thiols. To investigate the mode of action and stress response caused by allicin in S. aureus, we analyzed the transcriptome signature, the targets for S-thioallylation in the proteome and the changes in the bacillithiol (BSH) redox potential (EBSH) under allicin stress. Allicin caused a strong thiol-specific oxidative and sulfur stress response and protein damage as revealed by the induction of the PerR, HypR, QsrR, MhqR, CstR, CtsR, HrcA and CymR regulons in the RNA-seq transcriptome. Allicin also interfered with metal and cell wall homeostasis and caused induction of the Zur, CsoR and GraRS regulons. Brx-roGFP2 biosensor measurements revealed a strongly increased EBSH under allicin stress. In the proteome, 57 proteins were identified with S-thioallylations under allicin treatment, including translation factors (EF-Tu, EF-Ts), metabolic and redox enzymes (AldA, GuaB, Tpx, KatA, BrxA, MsrB) as well as redox-sensitive MarR/SarA-family regulators (MgrA, SarA, SarH1, SarS). Phenotype and biochemical analyses revealed that BSH and the HypR-controlled disulfide reductase MerA are involved in allicin detoxification in S. aureus. The reversal of protein S-thioallylation was catalyzed by the Brx/BSH/YpdA pathway. Finally, the BSSB reductase YpdA was shown to use S-allylmercaptobacillithiol (BSSA) as substrate to regenerate BSH in S. aureus. In conclusion, allicin results in an oxidative shift of EBSH and protein S-thioallylation, which can be reversed by YpdA and the Brx/BSH/YpdA electron pathways in S. aureus to regenerate thiol homeostasis.

Conservation of bacterial protein synthesis machinery: initiation and elongation in Mycobacterium smegmatis.

Most of our understanding of ribosome function is based on experiments utilizing translational components from Escherichia coli. It is not clear to which extent the details of translation mechanisms derived from this single organism are true for all bacteria. Here we investigate translation factor-dependent reactions of initiation and elongation in a reconstituted translation system from a Gram-positive bacterium Mycobacterium smegmatis. This organism was chosen because mutations in rRNA have very different phenotypes in E. coli and M. smegmatis, and the docking site for translational GTPases, the L12 stalk, is extended in the ribosomes from M. smegmatis compared to E. coli. M. smegmatis genes coding for IF1, IF2, IF3, EF-G, and EF-Tu were identified by sequence alignments; the respective recombinant proteins were prepared and studied in a variety of biochemical and biophysical assays with M. smegmatis ribosomes. We found that the activities of initiation and elongation factors and the rates of elemental reactions of initiation and elongation of protein synthesis are remarkably similar with M. smegmatis and E. coli components. The data suggest a very high degree of conservation of basic translation mechanisms, probably due to coevolution of the ribosome components and translation factors. This work establishes the reconstituted translation system from individual purified M. smegmatis components as an alternative to that from E. coli to study the mechanisms of translation and to test the action of antibiotics against Gram-positive bacteria.

MED: a new non-supervised gene prediction algorithm for bacterial and archaeal genomes.

BACKGROUND: Despite a remarkable success in the computational prediction of genes in Bacteria and Archaea, a lack of comprehensive understanding of prokaryotic gene structures prevents from further elucidation of differences among genomes. It continues to be interesting to develop new ab initio algorithms which not only accurately predict genes, but also facilitate comparative studies of prokaryotic genomes. RESULTS: This paper describes a new prokaryotic genefinding algorithm based on a comprehensive statistical model of protein coding Open Reading Frames (ORFs) and Translation Initiation Sites (TISs). The former is based on a linguistic "Entropy Density Profile" (EDP) model of coding DNA sequence and the latter comprises several relevant features related to the translation initiation. They are combined to form a so-called Multivariate Entropy Distance (MED) algorithm, MED 2.0, that incorporates several strategies in the iterative program. The iterations enable us to develop a non-supervised learning process and to obtain a set of genome-specific parameters for the gene structure, before making the prediction of genes. CONCLUSION: Results of extensive tests show that MED 2.0 achieves a competitive high performance in the gene prediction for both 5' and 3' end matches, compared to the current best prokaryotic gene finders. The advantage of the MED 2.0 is particularly evident for GC-rich genomes and archaeal genomes. Furthermore, the genome-specific parameters given by MED 2.0 match with the current understanding of prokaryotic genomes and may serve as tools for comparative genomic studies. In particular, MED 2.0 is shown to reveal divergent translation initiation mechanisms in archaeal genomes while making a more accurate prediction of TISs compared to the existing gene finders and the current GenBank annotation.

Transient kinetics, fluorescence, and FRET in studies of initiation of translation in bacteria.

Initiation of mRNA translation in prokaryotes requires the small ribosomal subunit (30S), initiator fMet-tRNA(fMet), three initiation factors, IF1, IF2, and IF3, and the large ribosomal subunit (50S). During initiation, the 30S subunit, in a complex with IF3, binds mRNA, IF1, IF2.GTP, and fMet-tRNA(fMet) to form a 30S initiation complex which then recruits the 50S subunit to yield a 70S initiation complex, while the initiation factors are released. Here we describe a transient kinetic approach to study the timing of elemental steps of 30S initiation complex formation, 50S subunit joining, and the dissociation of the initiation factors from the 70S initiation complex. Labeling of ribosomal subunits, fMet-tRNA(fMet), mRNA, and initiation factors with fluorescent reporter groups allows for the direct observation of the formation or dissociation of complexes by monitoring changes in the fluorescence of single dyes or fluorescence resonance energy transfer (FRET) between two fluorophores. Subunit joining was monitored by light scattering or by FRET between dyes attached to the ribosomal subunits. The kinetics of chemical steps, that is, GTP hydrolysis by IF2 and peptide bond formation following the binding of aminoacyl-tRNA to the 70S initiation complex, were measured by the quench-flow technique. The methods described here are based on results obtained with initiation components from Escherichia coli but can be adopted for mechanistic studies of initiation in other prokaryotic or eukaryotic systems.

Binding of mRNA to the bacterial translation initiation complex.

Translation initiation is a key step for regulating the synthesis of several proteins. In bacteria, translation initiation involves the interaction of the mRNA with the ribosomal small subunit. Additionally, translation initiation factors 1, 2, and 3, and the initiator tRNA, also assemble on the ribosomal small subunit and are essential for efficiently recruiting an mRNA for protein biosynthesis. In the following chapter, we describe fluorescence-based methods for studying the interaction of mRNA with the bacterial initiation complex. Model mRNAs with a covalently attached fluorescent probe showed an increase in fluorescence intensity when bound to the bacterial initiation complex. Utilizing the increase in fluorescence intensity upon mRNA binding to the bacterial initiation complex, we determined the equilibrium binding constants and the association and dissociation rate constants. These methods are important for quantitatively analyzing the effects of mRNA secondary structure and the role of the initiation factors in recruitment of mRNA by the bacterial initiation complex.