Molecular dissection of translation initiation factor IF2. Evidence for two structural and functional domains.

By means of limited proteolysis of Bacillus stearothermophilus initiation factor IF2 and genetic manipulation of its structural gene, infB, we have been able to produce (or hyperproduce) and purify two polypeptide fragments corresponding to two structurally and functionally separate domains of the protein. The first is the G-domain (approximately 41 kDa), which makes up the central part of the molecule and contains the conserved structural elements found in all GTP/GDP-binding sites of G-proteins. This domain is resistant to proteolysis in the presence of GTP or GDP, retains the capacity to interact with the 50 S subunit, binds weakly to the 30 S subunit, and displays ribosome-dependent GTPase activity with an approximately 2-fold higher Km for GTP and the same Vmax as compared with intact IF2. The second is the C-domain (approximately 24 kDa), which corresponds to the COOH-terminal part of IF2 and constitutes an extraordinarily compact domain containing the fMet-tRNA binding site of IF2. In spite of its negligible affinity for the ribosomes, the C-domain weakly stimulates the ribosomal binding of fMet-tRNA, presumably by affecting the conformation of the initiator tRNA molecule

Ribosomal selection of mRNAs with degenerate initiation triplets

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

Role of the ribosome‐associated protein PY in the cold‐shock response of E scherichia coli

Protein Y ( PY ) is an E scherichia coli cold‐shock protein which has been proposed to be responsible for the repression of bulk protein synthesis during cold adaptation. Here, we present in vivo and in vitro data which clarify the role of PY and its mechanism of action. Deletion of yfiA , the gene encoding protein PY , demonstrates that this protein is dispensable for cold adaptation and is not responsible for the shutdown of bulk protein synthesis at the onset of the stress, although it is able to partially inhibit translation. In vitro assays reveal that the extent of PY inhibition changes with different mRNA s and that this inhibition is related to the capacity of PY of binding 30S subunits with a fairly strong association constant, thus stimulating the formation of 70S monomers. Furthermore, our data provide evidence that PY competes with the other ribosomal ligands for the binding to the 30S subunits. Overall these results suggest an alternative model to explain PY function during cold shock and to reconcile the inhibition caused by PY with the active translation observed for some mRNA s during cold shock. E scherichia coli responds to cold stress by entering an acclimation phase during which protein synthesis slows down considerably with the exception of a specific set of genes (cold‐shock genes) whose expression is stimulated. In this article, we have investigated in vivo and in vitro the role of PY , a protein that is associated with the ribosome throughout the cold acclimation phase. Our data indicate that protein PY can affect translation initiation but is not responsible for turning off bulk protein synthesis during the cold stress.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/97494/1/mbo368-sup-0001-FigureS1-S3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/97494/2/mbo368.pd

High-affinity DNA binding sites for H-NS provide a molecular basis for selective silencing within proteobacterial genomes

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

Weakening the IF2-fMet-tRNA Interaction Suppresses the Lethal Phenotype Caused by GTPase Inactivation

Substitution of the conserved Histidine 448 present in one of the three consensus elements characterizing the guanosine nucleotide binding domain (IF2 G2) of Escherichia coli translation initiation factor IF2 resulted in impaired ribosome-dependent GTPase activity which prevented IF2 dissociation from the ribosome, caused a severe protein synthesis inhibition, and yielded a dominant lethal phenotype. A reduced IF2 affinity for the ribosome was previously shown to suppress this lethality. Here, we demonstrate that also a reduced IF2 affinity for fMet-tRNA can suppress this dominant lethal phenotype and allows IF2 to support faithful translation in the complete absence of GTP hydrolysis. These results strengthen the premise that the conformational changes of ribosome, IF2, and fMet-tRNA occurring during the late stages of translation initiation are thermally driven and that the energy generated by IF2-dependent GTP hydrolysis is not required for successful translation initiation and that the dissociation of the interaction between IF2 C2 and the acceptor end of fMet-tRNA, which represents the last tie anchoring the factor to the ribosome before the formation of an elongation-competent 70S complex, is rate limiting for both the adjustment of fMet-tRNA in a productive P site and the IF2 release from the ribosome

Translation initiation factor IF3: two domains, five functions, one mechanism?

Initiation factor IF3 contains two domains separated by a flexible linker. While the isolated N-domain displayed neither affinity for ribosomes nor a detectable function, the isolated C-domain, added in amounts compensating for its reduced affinity for 30S subunits, performed all activities of intact IF3, namely: (i) dissociation of 70S ribosomes; (ii) shift of 30S-bound mRNA from ‘stand-by’ to ‘P-decoding’ site; (iii) dissociation of 30S–poly(U)–NacPhe-tRNA pseudo- initiation complexes; (iv) dissociation of fMet-tRNA from initiation complexes containing mRNA with the non-canonical initiation triplet AUU (AUUmRNA); (v) stimulation of mRNA translation regardless of its start codon and inhibition of AUUmRNA translation at high IF3C/ribosome ratios. These results indicate that while IF3 performs all its functions through a C-domain–30S interaction, the N-domain function is to provide additional binding energy so that its fluctuating interaction with the 30S subunit can modulate the thermodynamic stability of the 30S–IF3 complex and IF3 recycling. The localization of IF3C far away from the decoding site and anticodon stem–loop of P-site-bound tRNA indicates that the IF3 fidelity function does not entail its direct contact with these structures