The translation initiation efficiency of a given mRNA is determined by its translation initiation region (TIR). mRNAs are selected into 30S initiation complexes according to the strengths of the secondary structure of the TIR, the pairing of the Shine-Dalgarno sequence with 16S rRNA, and the interaction between initiator tRNA and the start codon. Here, we show that the conversion of the 30S initiation complex into the translating 70S ribosome constitutes another important mRNA control checkpoint. Kinetic analysis reveals that 50S subunit joining and dissociation of IF3 are strongly influenced by the nature of the codon used for initiation and the structural elements of the TIR. Coupling between the TIR and the rate of 70S initiation complex formation involves IF3- and IF1-induced rearrangements of the 30S subunit, providing a mechanism by which the ribosome senses the TIR and determines the efficiency of translational initiation of a particular mRNA.
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.
M.Falconi and B.Colonna contributed equally to this workThe expression of plasmid-borne virF of Shigella encoding a transcriptional regulator of the AraC family, is required to initiate a cascade of events resulting in activation of several operons encoding invasion functions. H-NS, one of the main nucleoid-associated proteins, controls the temperature-dependent expression of the virulence genes by repressing the in vivo transcription of virF only below a critical temperature (~32°C). This temperature-dependent transcriptional regulation has been reproduced in vitro and the targets of H-NS on the virF promoter were identified as two sites centred around -250 and -1 separated by an intrinsic DNA curvature. H-NS bound cooperatively to these two sites below 32°C, but not at 37°C. DNA supercoiling within the virF promoter region did not influence H-NS binding but was necessary for the H-NS-mediated transcriptional repression. Electrophoretic analysis between 4 and 60°C showed that the virF promoter fragment, comprising the two H-NS sites, undergoes a specific and temperature-dependent conformational transition at~32°C. Our results suggest that this modification of the DNA target may modulate a cooperative interaction between H-NS molecules bound at two distant sites in the virF promoter region and thus represents the physical basis for the H-NS-dependent thermoregulation of virulence gene expression.
The global transcriptional regulator H-NS selectively silences bacterial genes associated with pathogenicity and responses to environmental insults. Although there is ample evidence that H-NS binds preferentially to DNA containing curved regions, we show here that a major basis for this selectivity is the presence of a conserved sequence motif in H-NS target transcriptons. We further show that there is a strong tendency for the H-NS binding sites to be clustered, both within operons and in genes contained in the pathogenicity-associated islands. In accordance with previously published findings, we show that these motifs occur in AT-rich regions of DNA. On the basis of these observations, we propose that H-NS silences extensive regions of the bacterial chromosome by binding first to nucleating high-affinity sites and then spreading along AT-rich DNA. This spreading would be reinforced by the frequent occurrence of the motif in such regions. Our findings suggest that such an organization enables the silencing of extensive regions of the genetic material, thereby providing a coherent framework that unifies studies on the H-NS protein and a concrete molecular basis for the genetic control of H-NS transcriptional silencing.
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