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Initiation factor IF3 is an essential protein that enhances the fidelity and speed of bacterial mRNA translation initiation. Here, we describe the dynamic interplay between IF3 domains and their alternative binding sites using pre-steady state kinetics combined with molecular modelling of available structures of initiation complexes. Our results show that IF3 accommodates its domains at velocities ranging over two orders of magnitude, responding to the binding of each 30S ligand. IF1 and IF2 promote IF3 compaction and the movement of the C-terminal domain (IF3C) towards the P site. Concomitantly, the N-terminal domain (IF3N) creates a pocket ready to accept the initiator tRNA. Selection of the initiator tRNA is accompanied by a transient accommodation of IF3N towards the 30S platform. Decoding of the mRNA start codon displaces IF3C away from the P site and rate limits translation initiation. 70S initiation complex formation brings IF3 domains in close proximity to each other prior to dissociation and recycling of the factor for a new round of translation initiation. Altogether, our results describe the kinetic spectrum of IF3 movements and highlight functional transitions of the factor that ensure accurate mRNA translation initiation. InnovatePeru [382-PNICP-PIBA-2014 and 297INNOVATEPERU-EC-2016 to P.M.]; Fondo Nacional de Desarrollo Cientifico, Tecnologico y de Innovacion Tecnologica [154-2017-Fondecyt and 0362019-Fondecyt-BM-INC.INV to P.M.]; FIRB Futuro in Ricerca [RBFR130VS5 001 to A.F.]; Italian Ministero dell'Istruzione, dell'Universita e della Ricerca (to A.F.); Part of the work on structural dynamics of the ribosome was supported by Russian Science Foundation [17-1401416 to A.L.K.]. Funding for open access: Universidad Peruana de Ciencias Aplicadas (Exp-03).

Pseudomonas aeruginosa is an opportunistic bacterial pathogen and a primary cause of nosocomial infection in humans. The rate of antibiotic resistance in P. aeruginosa is increasing worldwide leading to an unmet need for discovery of new chemical compounds distinctly different from present antimicrobials. Protein synthesis is an essential metabolic process and a validated target for the development of new antibiotics. Initiation factor 1 from P. aeruginosa (Pa-IF1) is the smallest of the three initiation factors that act to establish the 30S initiation complex during initiation of protein biosynthesis. Here we report the characterization and solution NMR structure of Pa-IF1. Pa-IF1 consists of a five-stranded β-sheet with an unusual extended β-strand at the C-terminus and one short α-helix arranged in the sequential order β1-β2-β3-α1-β4-β5. The structure adopts a typical β-barrel fold and contains an oligomer-binding motif. A cluster of basic residues (K39, R41, K42, K64, R66, R70, and R72) located on the surface of strands β4 and β5 near the short α-helix may compose the binding interface with the 30S subunit.

The transition of the 30S initiation complex (IC) to the translating 70S ribosome after 50S subunit joining provides an important checkpoint for mRNA selection during translation in bacteria. Here, we study the timing and control of reactions that occur during 70S IC formation by rapid kinetic techniques, using a toolbox of fluorescence-labeled translation components. We present a kinetic model based on global fitting of time courses obtained with eight different reporters at increasing concentrations of 50S subunits. IF1 and IF3 together affect the kinetics of subunit joining, but do not alter the elemental rates of subsequent steps of 70S IC maturation. After 50S subunit joining, IF2-dependent reactions take place independent of the presence of IF1 or IF3. GTP hydrolysis triggers the efficient dissociation of fMet-tRNAfMet from IF2 and promotes the dissociation of IF2 and IF1 from the 70S IC, but does not affect IF3. The presence of non-hydrolyzable GTP analogs shifts the equilibrium towards a stable 70S–mRNA–IF1–IF2–fMet-tRNAfMet complex. Our kinetic analysis reveals the molecular choreography of the late stages in translation initiation. Boehringen Ingelheim Fonds and the G¨ottingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences (to A.G.); Max Planck Society and grants of the Deutsche Forschungsgemeinschaft (to M.V.R.); Peruvian Programa Nacional de Innovaci ´on para la Competitividad y Productividad [382-PNICP-PIBA-2014 (to P.M.)]. Funding for open access charge: Max Planck Society. Revisión por pares

Genes consist of multiple sequence elements that together encode the functional product and regulate its expression. Despite their fundamental importance, there are few freely available diagrams of gene structure. Presented here are two figures that summarise the different structures found in eukaryotic and prokaryotic genes. Common gene structural elements are colour-coded by their function in regulation, transcription, or translation.

To assess the influence of degenerate initiation triplets on mRNA recruitment by ribosomes, five mRNAs identical but for their start codon (AUG, GUG, UUG, AUU and AUA) were offered to a limiting amount of ribosomes, alone or in competition with an identical AUGmRNA bearing a mutation conferring different electrophoretic mobility to the product. Translational efficiency and competitiveness of test mRNAs toward this AUGmRNA were determined quantifying the relative amounts of the electrophoretically separated wt and mutated products synthesized in vitro and found to be influenced to different extents by the nature of their initiation triplet and by parameters such as temperature and nutrient availability in the medium. The behaviors of AUAmRNA, UUGmRNA and AUGmRNA were the same between 20 and 40°C whereas the GUG and AUUmRNAs were less active and competed poorly with the AUGmRNA, especially at low temperature. Nutrient limitation and preferential inhibition by ppGpp severely affected activity and competitiveness of all mRNAs bearing non-AUG starts, the UUGmRNA being the least affected. Overall, our data indicate that beyond these effects exclusively due to the degenerate start codons within an optimized translational initiation region, an important role is played by the context in which the rare start codons are present.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen and a primary cause of nosocomial infection in humans. The rate of antibiotic resistance in P. aeruginosa is increasing worldwide leading to an unmet need for discovery of new chemical compounds distinctly different from present antimicrobials. Protein synthesis is an essential metabolic process and a validated target for the development of new antibiotics. Initiation factor 1 from P. aeruginosa (Pa-IF1) is the smallest of the three initiation factors that act to establish the 30S initiation complex during initiation of protein biosynthesis. Here we report the characterization and solution NMR structure of Pa-IF1. Pa-IF1 consists of a five-stranded β-sheet with an unusual extended β-strand at the C-terminus and one short α-helix arranged in the sequential order β1-β2-β3-α1-β4-β5. The structure adopts a typical β-barrel fold and contains an oligomer-binding motif. A cluster of basic residues (K39, R41, K42, K64, R66, R70, and R72) located on the surface of strands β4 and β5 near the short α-helix may compose the binding interface with the 30S subunit.

Genetic selection has been used to isolate second-site suppressors of a defective cold-sensitive initiation factor I (IF1) R69L mutant of Escherichia coli. The suppressor mutants specifically map t ...

Protein synthesis is principally regulated at the initiation stage (rather than during elongation or termination), allowing rapid, reversible and spatial control over gene expression. Progress over recent years in determining the structures and activities of initiation factors, and in mapping their interactions within ribosomal initiation complexes, has significantly advanced our understanding of the complex translation initiation process. These developments have provided a solid foundation for studies of regulation of initiation by mechanisms that include modulation of the activity of initiation factors (which affects almost all scanning-dependent initiation), or via sequence-specific RNA-binding proteins and microRNAs (which thus impact individual mRNAs).

The mitochondrial F1F0 ATP synthase is responsible for the majority of ATP production in mammals and does this through a rotary catalytic mechanism. Studies show that the F1F0 ATP synthase can switch to an ATP hydrolase, and this occurs under conditions seen during myocardial ischemia. This ATP hydrolysis causes wasting of ATP that does not produce work. The degree of ATP inefficiently hydrolyzed during ischemia may be as high as 50-90% of the total. A naturally occurring, reversible inhibitor (IF-1) of the hydrolase activity is in the mitochondria, and it has a pH optimum of 6.8. Based on studies with the nonselective (inhibit both synthase and hydrolase activity) inhibitors aurovertin B and oligomycin B reduce the rate of ATP depletion during ischemia, showing that IF-1 does not completely block hydrolase activity. Oligomycin and aurovertin cannot be used for treating myocardial ischemia as they will reduce ATP production in healthy tissue. We generated a focused structure-activity relationship, and several compounds were identified that selectively inhibited the F1F0 ATP hydrolase activity while having no effect on synthase function. One compound, BMS-199264 had no effect on F1F0 ATP synthase function in submitochondrial particles while inhibiting hydrolase function, unlike oligomycin that inhibits both. BMS-199264 selectively inhibited ATP decline during ischemia while not affecting ATP production in normoxic and reperfused hearts. BMS-191264 also reduced cardiac necrosis and enhanced the recovery of contractile function following reperfusion. These data also suggest that the reversal of the synthase and hydrolase activities is not merely a chemical reaction run in reverse.

International audience; Translation initiation, the rate-limiting step of the universal process of protein synthesis, proceeds through sequential, tightly regulated steps. In bacteria, the correct messenger RNA start site and the reading frame are selected when, with the help of initiation factors IF1, IF2 and IF3, the initiation codon is decoded in the peptidyl site of the 30S ribosomal subunit by the fMet-tRNA(fMet) anticodon. This yields a 30S initiation complex (30SIC) that is an intermediate in the formation of the 70S initiation complex (70SIC) that occurs on joining of the 50S ribosomal subunit to the 30SIC and release of the initiation factors. The localization of IF2 in the 30SIC has proved to be difficult so far using biochemical approaches, but could now be addressed using cryo-electron microscopy and advanced particle separation techniques on the basis of three-dimensional statistical analysis. Here we report the direct visualization of a 30SIC containing mRNA, fMet-tRNA(fMet) and initiation factors IF1 and GTP-bound IF2. We demonstrate that the fMet-tRNA(fMet) is held in a characteristic and precise position and conformation by two interactions that contribute to the formation of a stable complex: one involves the transfer RNA decoding stem which is buried in the 30S peptidyl site, and the other occurs between the carboxy-terminal domain of IF2 and the tRNA acceptor end. The structure provides insights into the mechanism of 70SIC assembly and rationalizes the rapid activation of GTP hydrolysis triggered on 30SIC-50S joining by showing that the GTP-binding domain of IF2 would directly face the GTPase-activated centre of the 50S subunit.

The mechanism of translation in eubacteria and organelles is thought to be similar. In eubacteria, the three initiation factors IF1, IF2, and IF3 are vital. Although the homologs of IF2 and IF3 are found in mammalian mitochondria, an IF1 homolog has never been detected. Here, we show that bovine mitochondrial IF2 (IF2(mt)) complements E. coli containing a deletion of the IF2 gene (E. coli DeltainfB). We find that IF1 is no longer essential in an IF2(mt)-supported E. coli DeltainfB strain. Furthermore, biochemical and molecular modeling data show that a conserved insertion of 37 amino acids in the IF2(mt) substitutes for the function of IF1. Deletion of this insertion from IF2(mt) supports E. coli for the essential function of IF2. However, in this background, IF1 remains essential. These observations provide strong evidence that a single factor (IF2(mt)) in mammalian mitochondria performs the functions of two eubacterial factors, IF1 and IF2.

Aim: Wolbachia is a promising antifilarial chemotherapeutic target. Translation initiation factor-1 (Tl IF-1) is an essential factor in prokaryotes. Functional characterization of Wolbachia’s novel proteins/enzymes is necessary for the development of adulticidal drugs. Materials & methods: Mutant, Wol Tl IF-1 R45D was constructed by site directed mutagenesis. Fluorimetry and size exclusion chromatography were used to determine the biophysical characteristics. Mobility shift assay and fluorescence resonance energy transfer were used to investigate the functional aspect of Wol Tl IF-1 with its mutant. Results: Both wild and mutant were in monomeric native conformations. Wild exhibits nonspecific binding with ssRNA/ssDNA fragments under electrostatic conditions and showed annealing and displacement of RNA strands in comparison to mutant. Conclusion: Point mutation impaired RNA chaperone activity of the mutant and its interaction with nucleotides.

According to the standard model of bacterial translation initiation, the small ribosomal 30S subunit binds to the initiation site of an mRNA with the help of three initiation factors (IF1-IF3). Here, we describe a novel type of initiation termed "70S-scanning initiation," where the 70S ribosome does not necessarily dissociate after translation of a cistron, but rather scans to the initiation site of the downstream cistron. We detailed the mechanism of 70S-scanning initiation by designing unique monocistronic and polycistronic mRNAs harboring translation reporters, and by reconstituting systems to characterize each distinct mode of initiation. Results show that 70S scanning is triggered by fMet-tRNA and does not require energy; the Shine-Dalgarno sequence is an essential recognition element of the initiation site. IF1 and IF3 requirements for the various initiation modes were assessed by the formation of productive initiation complexes leading to synthesis of active proteins. IF3 is essential and IF1 is highly stimulating for the 70S-scanning mode. The task of IF1 appears to be the prevention of untimely interference by ternary aminoacyl (aa)-tRNA•elongation factor thermo unstable (EF-Tu)•GTP complexes. Evidence indicates that at least 50% of bacterial initiation events use the 70S-scanning mode, underscoring the relative importance of this translation initiation mechanism.

During initiation of bacterial protein synthesis, messenger RNA and fMet-tRNAfMet bind to the 30S ribosomal subunit together with initiation factors IF1, IF2, and IF3. Docking of the 30S preinitiation complex to the 50S ribosomal subunit results in a peptidyl-transfer competent 70S ribosome. Initiation with an elongator tRNA may lead to frameshift and an aberrant N-terminal sequence in the nascent protein. We show how the occurrence of initiation errors is minimized by (1) recognition of the formyl group by the synergistic action of IF2 and IF1, (2) uniform destabilization of the binding of all tRNAs to the 30S subunit by IF3, and (3) an optimal distance between the Shine-Dalgarno sequence and the initiator codon. We suggest why IF1 is essential for E. coli, discuss the role of the G-C base pairs in the anticodon stem of some tRNAs, and clarify gene expression changes with varying IF3 concentration in the living cell.

The influence in vivo of mutated forms of translation initiation factor (IF1) on the expression of the lacZ or 3A′ reporter genes, with different initiation and/or +2 codons, has been investigated. Reporter gene expression in these infA(IF1) mutants is similar to the wild-type strain. The results do not support the longstanding hypothesis that IF1 could perform discriminatory functions while blocking the aminoacyl-tRNA acceptor site (A-site) of the ribosome. One cold-sensitive IF1 mutant shows a general overexpression, in particular at low temperatures, of both reporter genes at the protein but not mRNA level.

Uneven distribution of plasmid-based expression vectors to daughter cells during bacterial cell division results in an increasing proportion of plasmid free cells during growth. This is a major industrial problem leading to reduction of product yields and increased production costs during large-scale cultivation of vector-carrying bacteria. For this reason, a selection must be provided that kills the plasmid free cells. The most conventional method to obtain this desired selection is to insert some gene for antibiotic resistance in the plasmid and then grow the bacteria in the presence of the corresponding antibiotic. We describe here a host/plasmid Escherichia coli system with a totally stable plasmid that can be maintained without the use of antibiotic selection. The plasmid is maintained, since it carries the small essential gene infA (coding for translation initiation factor 1, IF1) in an E. coli strain that has been deleted for its chromosomal infA gene. As a result only plasmid carrying cells can grow, making the strain totally dependent on the maintenance of the plasmid. A selection based on antibiotics is thus not necessary during cultivation, and no antibiotic-resistance genes are present neither in the final strain nor in the final plasmid. Plasmid-free cells do not accumulate even after an extended period of continuous growth. Growth rates of the control and the plasmid harboring strains are indistinguishable from each other in both LB and defined media. The indicated approach can be used to modify existing production strains and plasmids to the described concept. The infA based plasmid stability system should eliminate industrial cultivation problems caused by the loss of expression vector and use of antibiotics in the cultivation medium. Also environmental problems caused by release of antibiotics and antibiotic resistance genes, that potentially can give horizontal gene transfer between bacterial populations, are eliminated.

Protein synthesis is one of the most complex cellular processes, involving numerous translation components that interact in multiple sequential steps. The most complex stage in protein synthesis is the initiation process. It involves initiation factor-mediated assembly of a 40S ribosomal subunit and initiator tRNA into a 48S initiation complex at the initiation codon of an mRNA and subsequent joining of a 60S ribosomal subunit to form a translationally active 80S ribosome. The basal set of factors required for translation initiation has been determined, and biochemical, genetic, and structural studies are now beginning to reveal details of their individual functions in this process. The mechanism of translation initiation has also been found to be influenced significantly by structural properties of the 5' and 3' termini of individual mRNAs. This review describes some of the major developments in elucidating molecular details of the mechanism of initiation that have occurred over the last decade.

Initiation of protein biosynthesis in bacteria requires three initiation factors: initiation factor 1, initiation factor 2 and initiation factor 3. The mechanism by which initiation factors form the 70S initiation complex with initiator fMet-tRNA(fMet) interacting with the initiation codon in the ribosomal P site and the second mRNA codon exposed in the A site is not yet understood. Here, we present a model for the function of initiation factors 1 and 2 that is based on the analysis of sequence homologies, biochemical evidence and the present knowledge of the three-dimensional structures of translation factors and ribosomes. The model predicts that initiation factors 1 and 2 interact with the ribosomal A site mimicking the structure of the elongation factor G. We present data that extend the mimicry hypothesis to initiation factors 1 and 2, originally postulated for the aminoacyl-tRNA x elongation factor Tu x GTP ternary complex, elongation factor G and release factors.

Translation generally initiates with the AUG codon. While initiation at GUG and UUG is permitted in prokaryotes (Archaea and Bacteria), cases of CUG initiation were recently reported in human cells. The varying stringency in translation initiation between eukaryotic and prokaryotic domains largely stems from a fundamental problem for the ribosome in recognizing a codon at the peptidyl-tRNA binding site. Initiation factors specific to each domain of life evolved to confer stringent initiation by the ribosome. The mechanistic basis for high accuracy in eukaryotic initiation is described based on recent findings concerning the role of the multifactor complex (MFC) in this process. Also discussed are whether non-AUG initiation plays any role in translational control and whether start codon accuracy is regulated in eukaryotes.

A mutation in the infA gene encoding initiation factor 1 (IF1) gives rise to a cold-sensitive phenotype. An Escherichia coli strain with this mutation was used as a tool to select for second-site suppressors that compensate for the cold sensitivity and map specifically to rRNA. Several suppressor mutants with altered 16S rRNA that partially restore growth of an IF1 mutant strain in the cold were isolated and characterized. Suppressor mutations were found in helix (h)18, h32, h34 and h41 in 16S rRNA. These mutations are not clustered to any particular region in 16S rRNA and none overlap previously reported sites of interaction with IF1. While the isolated suppressors are structurally diverse, they are functionally related because all affect ribosomal subunit association in vivo. Furthermore, in vitro subunit-association experiments indicate that most of the suppressor mutations directly influence ribosomal subunit association even though none of these are confined to any of the known intersubunit bridges. These results are consistent with the model that IF1 is an rRNA chaperone that induces large-scale conformational changes in the small ribosomal subunit, and as a consequence modulates initiation of translation by affecting subunit association.

Genetic selection has been used to isolate second-site suppressors of a defective cold-sensitive initiation factor I (IF1) R69L mutant of Escherichia coli. The suppressor mutants specifically map to a single rRNA operon on a plasmid in a strain with all chromosomal rRNA operons deleted. Here, we describe a set of suppressor mutations that are located in the processing stem of precursor 23S rRNA. These mutations interfere with processing of the 23S rRNA termini. A lesion of RNase III also suppresses the cold sensitivity. Our results suggest that the mutant IF1 strain is perturbed at the level of ribosomal subunit association, and the suppressor mutations partially compensate for this defect by disrupting rRNA maturation. These results support the notion that IF1 is an RNA chaperone and that translation initiation is coupled to ribosomal maturation.

Initiation is a critical step in translation, during which the ribosome lands on the start codon and sets the correct reading frame for mRNA decoding. The rate and efficiency of translation are largely determined by initiation, which is therefore the preferred target of translation regulation mechanisms. Initiation has incurred an extensive evolutionary divergence among the primary domains of cell descent. The Archaea, albeit prokaryotes, have an initiation mechanism and apparatus more complex than those of the Bacteria; the molecular details of archaeal initiation are just beginning to be unravelled. The most notable aspects of archaeal initiation are the presence of two, perhaps three, distinct mechanisms for mRNA–ribosome interaction and the presence of a relatively large set of IFs (initiation factors), several of which are shared exclusively with the Eukarya. Among these, the protein termed a/eIF2 (archaeal/eukaryotic IF2) and aIF6 (archaeal IF6) are of special interest, since they appear to play key regulatory roles in the Eukarya. Studies of the function of these factors in Archaea have uncovered new features that will help to elucidate their conserved and domain-specific functions.

Translation initiation is a critical step in protein synthesis. Previously, two major mechanisms of initiation were considered as essential: prokaryotic, based on SD interaction; and eukaryotic, requiring cap structure and ribosomal scanning. Although discovered decades ago, cap-independent translation has recently been acknowledged as a widely spread mechanism in viruses, which may take place in some cellular mRNA translations. Moreover, it has become evident that translation can be initiated on the leaderless mRNA in all three domains of life. New findings demonstrate that other distinguishable types of initiation exist, including SD-independent in Bacteria and Archaea, and various modifications of 5′ end-dependent and internal initiation mechanisms in Eukarya. Since translation initiation has developed through the loss, acquisition, and modification of functional elements, all of which have been elevated by competition with viral translation in a large number of organisms of different complexity, more variation in initiation mechanisms can be anticipated.

Initiation of translation is a complicated process involving numerous accessory factors whose functions remain incompletely understood. Bacterial ribosomal protein S1 is known to contain a repeated sequence motif (S1-RM), also found in polynucleotide phosphorylase, that is thought to be involved in binding to RNA. Using the technique of profile analysis, the S1-RM can also be found in bacterial and chloroplast translation initiation factor IF-1 sequences, and in the sequences of eukaryotic translation initiation factor eIF-2α chains. The significance of the similarity of the sequences is very high suggesting that the occurrence of the S1-RM in these diverse proteins represents homology. The similarity of S1 to IF-1 further suggests that S1 has evolved from an IF-1 like ancestor, and therefore that the two proteins have a similar or competitive function. The most obvious common function of the proteins containing the S1-RM seems to be RNA binding, suggesting that IF-1 and eIF-2α may bind to RNA.

In Escherichia coli, temperature downshift elicits cold shock response, which is characterized by induction of cold shock proteins. CspA, the major cold shock protein of E. coli, helps cells to acclimatize to low temperature by melting the secondary structures in nucleic acids and acting as a transcription antiterminator. CspA and its homologues contain the cold shock domain and belong to the oligomer binding protein family, which also includes S1 domain proteins such as IF1. Structural similarity between IF1 and CspA homologues suggested a functional overlap between these proteins. Indeed IF1 can melt secondary structures in RNA and acts as transcription antiterminator in vivo and in vitro. Here, we show that in spite of having these critical activities, IF1 does not complement cold-sensitivity of a csp quadruple deletion strain. DNA microarray analysis shows that overproduction of IF1 and Csp leads to changes in expression of different sets of genes. Importantly, several genes which were previously shown to require Csp proteins for their expression at low temperature did not respond to IF1. Moreover, in vitro, we show that a transcription terminator responsive to Csp does not respond to IF1. Our results suggest that Csp proteins and IF1 have different sets of target genes as they may be suppressing the function of different types of transcription termination elements in specific genes.

International audience; Tight recognition of codon-anticodon pairings by the ribosome ensures the accuracy and fidelity of protein synthesis. In eubacteria, translational surveillance and ribosome rescue are performed by the 'tmRNA-SmpB' system (transfer messenger RNA-small protein B). Remarkably, entry and accommodation of aminoacylated-tmRNA into stalled ribosomes occur without a codon-anticodon interaction but in the presence of SmpB. Here, we show that within a stalled ribosome, SmpB interacts with the three universally conserved bases G530, A1492 and A1493 that form the 30S subunit decoding centre, in which canonical codon-anticodon pairing occurs. The footprints at positions A1492 and A1493 of a small decoding centre, as well as on a set of conserved SmpB amino acids, were identified by nuclear magnetic resonance. Mutants at these residues display the same growth defects as for DeltasmpB strains. The SmpB protein has functional and structural similarities with initiation factor 1, and is proposed to be a functional mimic of the pairing between a codon and an anticodon.

Bacillus stearothermophilus infA encoding translation initiation factor IF1 was cloned and expressed in Escherichia coli and its transcript and protein product characterized. Although the functional properties of B. stearothermophilus and E. coli IF1, compared in several translational tests in the presence of both homologous and heterologous components, are not entirely identical, the two proteins are interchangeable in an in vitro translational system programmed with a natural mRNA. The availability of purified B. stearothermophilus IF1 now allows us to analyze the translation initiation pathway using efficient in vitro tests based entirely on purified components derived from this thermophilic Gram-positive bacterium.

We considered, on a global scale, the relationship between the predicted fraction of protein disorder and the RNA and protein expression in Escherichia coli. Fraction of protein disorder correlated positively with both measured RNA expression levels of E. coli genes in three different growth media and with predicted abundance levels of E. coli proteins. Though weak, the correlation was highly significant. Correlation of protein disorder with RNA expression did not depend on the growth rate of E. coli cultures and was not caused by a small subset of genes showing exceptionally high concordance in their disorder and expression levels. Global analysis was complemented by detailed consideration of several groups of proteins.

We demonstrate that ribosomes containing a messenger RNA (mRNA) with a strong Shine-Dalgarno sequence are rapidly split into subunits by initiation factors 1 (IF1) and 3 (IF3), but slowly split by ribosome recycling factor (RRF) and elongation factor G (EF-G). Post-termination-like (PTL) ribosomes containing mRNA and a P-site-bound deacylated transfer RNA (tRNA) are split very rapidly by RRF and EF-G, but extremely slowly by IF1 and IF3. Vacant ribosomes are split by RRF/EF-G much more slowly than PTL ribosomes and by IF1/IF3 much more slowly than mRNA-containing ribosomes. These observations reveal complementary splitting of different ribosomal complexes by IF1/IF3 and RRF/EF-G, and suggest the existence of two major pathways for ribosome splitting into subunits in the living cell. We show that the identity of the deacylated tRNA in the PTL ribosome strongly affects the rate by which it is split by RRF/EF-G and that IF3 is involved in the mechanism of ribosome splitting by IF1/IF3 but not by RRF/EF-G. With support from our experimental data, we discuss the principally different mechanisms of ribosome splitting by IF1/IF3 and by RRF/EF-G.

Bacterial translation initiation factor IF1 is homologous to archaeal aIF1A and eukaryal eIF1A, which form a complex with their homologous IF2-like factors (aIF5B and eIF5B respectively) during initiation of protein synthesis. A similar IF1-IF2 interaction is assumed to occur in all bacteria and supported by cross-linking data and stabilization of the 30S-IF2 interaction by IF1. Here we compare Escherichia coli IF1 with thermophilic factors from Bacillus stearothermophilus and Thermus thermophilus. All three IF1s are structurally similar and functionally interchangeable in vivo and in vitro. However, the thermophilic factors do not stimulate ribosomal binding of IF2DeltaN, regardless of 30S subunits and IF2 origin. We conclude that an IF1-IF2 interaction is not universally conserved and is not essential for cell survival.

Escherichia coli infA is transcribed from two promoters, P1 and P2, into a longer and a shorter mRNA encoding translation initiation factor IF1. Although P1 is intrinsically stronger than P2, the shorter half-life of its transcripts causes the steady-state level of the P2 transcript to be substantially higher than that of P1 during growth at 37 degrees C. After cold-shock, de novo transcription and translation of infA contribute to the transient increase of the IF1/ribosomes ratio, which is partially responsible for translational bias consisting in the preferential translation of cold-shock mRNAs in the cold. Cold-stress induction of infA expression is mainly due to the high activity of P1 at low temperature, which is further increased by transcriptional stimulation by CspA and by an increased transcript stability. Furthermore, the longer infA mRNA originating from P1 is preferentially translated at low temperature by the translational machinery of cold-shocked cells. The increased level of IF1 during cold adaptation is essential for overcoming the higher stability of the 70S monomers at low temperature and for providing a sufficient pool of dissociated 30S subunits capable of initiating translation.

Bacterial translation initiation factor IF1 is an S1 domain protein that belongs to the oligomer binding (OB) fold proteins. Cold shock domain (CSD)-containing proteins such as CspA (the major cold shock protein of Escherichia coli ) and its homologues also belong to the OB fold protein family. The striking structural similarity between IF1 and CspA homologues suggests a functional overlap between these proteins. Certain members of the CspA family of cold shock proteins act as nucleic acid chaperones: they melt secondary structures in nucleic acids and act as transcription antiterminators. This activity may help the cell to acclimatize to low temperatures, since cold-induced stabilization of secondary structures in nascent RNA can impede transcription elongation. Here we show that the E. coli translation initiation factor, IF1, also has RNA chaperone activity and acts as a transcription antiterminator in vivo and in vitro. We further show that the RNA chaperone activity of IF1, although critical for transcription antitermination, is not essential for its role in supporting cell growth, which presumably functions in translation. The results thus indicate that IF1 may participate in transcription regulation and that cross talk and/or functional overlap may exist between the Csp family proteins, known to be involved in transcription regulation at cold shock, and S1 domain proteins, known to function in translation.

Initiation factor 1 (IF1) is an essential protein in Escherichia coli involved in the initiation step of protein synthesis. The protein level of IF1 increases when E. coli cells are subjected to cold shock, however, it remains unclear as to how this increase occurs. The infA gene encoding IF1 contains two promoters, the distal P1 and the proximal P2 promoter. In this study, we found that infA mRNA was greatly increased, and that this increase resulted from transcriptional activation of P1, not P2, during cold shock although stability of transcripts from both promoters concomitantly increased.

Translation is one stage of protein synthesis in which messenger ribonucleic acid (mRNA) acts as a template for the synthesis of a polypeptide chain; it consists of four phases: initiation, elongation, termination and ribosome recycling. Initiation of protein synthesis, entailing ribosomal recognition of the mRNA start codon and the setting of the correct reading frame, is the rate-limiting step of translation and the main target of translation regulation in all cells. However, the mechanism and molecular machinery for initiation have diverged in the primary domains of life: the Bacteria, the Archaea and the Eukarya (eukaryotes). In bacteria, translation initiation is relatively simple, while in eukaryotes is complex and requires more components. In archaea, despite their prokaryotic phenotype, the machinery for protein synthesis initiation is much more elaborated than in bacteria and presents intriguing similarities with the corresponding eukaryotic process. The features of translational initiation in archaea, bacteria and eukaryotes are reviewed, highlighting the divergent and common aspects of this important cellular process in the three domains of life. Key concepts Initiation is the first of four steps of the translation cycle. During initiation the small ribosomal subunit interacts with the messenger ribonucleic acid (mRNA) and finds the right start codon, thereby setting the correct reading frame for decoding. In all cells, translation initiation is assisted and modulated by several proteins called translation initiation factors (IFs). The most important roles of the IFs are to select the proper initiator transfer RNA, to adjust it correctly in the ribosomal P site, to ensure the accurate selection of the start codon and to prevent the premature joining of the large ribosomal subunit to the pre-initiation complex. Initiation is the rate-limiting step of translation. It is the efficiency whereby an mRNA is decoded and the amount of final product is largely determined at the initiation level. In all cells, the principal mechanisms of translational control act at the level of initiation, usually targeting certain IFs that play essential roles in the initiation process. The initiation step of translation has incurred a marked evolutionary divergence. The mechanism and components of initiation differ in several aspects in the three primary domains of life, that is the Bacteria, the Archaea and the Eukaryotes. The Bacteria have the simplest machinery for translation initiation. However, this is not because they are prokaryotes. The other prokaryotic domain, the Archaea, has a fairly complex machinery for initiation, resembling that of eukaryotes, which have the most complex initiation process. Keywords: translation; initiation; mRNA; ribosome; RNA structure

To initiate protein synthesis, a ribosome with bound initiator methionyl-tRNA must be assembled at the start codon of an mRNA. This process requires the coordinated activities of three translation initiation factors (IF) in prokaryotes and at least 12 translation initiation factors in eukaryotes (eIF). The factors eIF1A and eIF5B from eukaryotes show extensive amino acid sequence similarity to the factors IF1 and IF2 from prokaryotes. By a combination of two-hybrid, coimmunoprecipitation, and in vitro binding assays eIF1A and eIF5B were found to interact directly, and the eIF1A binding site was mapped to the C-terminal region of eIF5B. This portion of eIF5B was found to be critical for growth in vivo and for translation in vitro. Overexpression of eIF1A exacerbated the slow-growth phenotype of yeast strains expressing C-terminally truncated eIF5B. These findings indicate that the physical interaction between the evolutionarily conserved factors eIF1A and eIF5B plays an important role in translation initiation, perhaps to direct or stabilize the binding of methionyl-tRNA to the ribosomal P site.

Evidence is presented that suggests the formation of a complex between polypeptide chain-initiation factor IF 2, GTP, and fMet-tRNA f . This complex transfers both fMet-tRNA f and GTP to 30S ribosomal subunits in the presence of ApUpG and initiation factor IF 1. The resultant 30S initiation complex reacts with 50S subunits to form a 70S initiation complex. fMet-tRNA f in this 70S complex reacts with puromycin to form fMet-puromycin. These results suggest that [IF 2, GTP, fMet-tRNA f ] is an intermediate in the initiation of protein synthesis in Escherichia coli .

Discrimination between eukaryotic and prokaryotic, and formylated and non-formylated, initiator tRNAs by eukaryotic initiation factor EIF-3

A method is presented for the detection in crude lysates of subnanogram amounts of proteins covalently bound to a specific nucleic acid sequence. The sensitivity of this method enabled us to study proteins cross-linked to specific DNA and mRNA sequences by irradiation of intact Escherichia coli cells with ultraviolet light. Among the proteins cross-linked to pBR322 DNA, the single strand binding protein, the HU-proteins, and the RNA polymerase beta and sigma subunits were present. Some, but not all proteins were cross-linked to 5-bromodeoxyuridine-substituted DNA more efficiently than to normal DNA. Ribosomal protein S1 is by far the most prominent protein cross-linked to mRNAs. Among the proteins cross-linked in smaller amounts to mRNAs are translation initiation factor IF 1, and at least six proteins of the 30 S ribosomal subunit, among which is S21. No 50 S proteins, nor IF-2, IF-3 or any of the elongation factors could be detected. Some UV-induced nucleic acid-protein cross-links were found to be heat-labile. It is concluded that the method employed may be used to compare the proteins interacting with different mRNAs, as well as single-copy DNA sequences from bacteria and eucaryotes with low complexity genomes.

By means of exchange studies, in which 3H-labelled 50-S subunits and unlabelled 70-S ribosomes from Escherichia coli MRE 600 were used, it has been demonstrated that the 30-S subunit is the only target for IF-3 in the dissociation of 70-S ribosomes. The interference of IF-3 with the dynamic equilibrium of 70-S in equilibrium 50-S + 30-S occurs by binding of the factor to the 30-S subunit. The 30-S-IF-3 complex in impaired in the association reaction, which implies that IF-3 is acting as an anti-association factor. The action of IF-1 is two-fold. Firstly IF-1 increases the rate of exhcange of the ribosomal subparticles in the 70-S ribosome without changing the position of the equilibrium. Thus the spontaneous equilibrium is attained more rapidly in the presence of IF-1. This kinetic effect of IF-1 is also demonstrated in the IF-3-mediated dissociation of 70-S ribosomes. Secondly IF-1 is able to increase the IF-3-mediated dissociation. It seems likely that the explanation for the latter phenomenon must be sought in the binding of IF-1 to 70-S ribosomes, resulting in a loosening of the ribosomes structure, as well as to 30-S. IF-3 complex, thaereby slowing down the association reactions of the subunits.

Specific in situ cleavage of 16S rRNA of E. coli has been accomplished by in vitro treatment of 70S ribosomes ("tight couples") with the bacteriocin cloacin DF13. The defective ribosomes, which have fully lost their ability to sustain polypeptide synthesis, are still able to form initiation on complexes with MS2 RNA, but the kinetics are altered. This is apparently due to an improper functioning of initiation factor IF-1, for the defective ribosomal couples respond normally to dissociation by IF-3 but the dissociation is not stimulated by IF-1. The initiation complexes formed with defective ribosomes are fully reactive with puromycin. Their ability to bind alanyl-tRNA is reduced by about 50% at all concentrations of elongation factor Tu studied. Cleavage of the 16S rRNA, not the release of the terminal fragment from the ribosome, causes the block of protein synthesis and the aberrations observed during initiation and elongation.