The how and Y of cold shock

Cold shock occurs when bacteria are exposed to sudden downshifts in temperature, and is characterized by the expression of a well-defined set of proteins, which include helicases, nucleases and a plethora of nucleic acid−binding proteins such as the so-called cold shock family of proteins (Csps)^{2, 3, 4}. In contrast to stress conditions like heat shock, no cold-specific factor has so far been identified, suggesting that the cold shock response is predominantly post-transcriptional². Consistent with this notion, many of the proteins up-regulated in E. coli during cold shock are associated with the translational apparatus, such as the trigger factor, a ribosome-binding protein chaperone, CsdA, an ATP-dependent RNA helicase-like protein, RbfA, an RNA-binding protein implicated in ribosome assembly, and the translation initiation factors IF1 and IF3 (refs. 2, 3, 4).

The inhibition of translation owing to temperature decrements has been generally thought to result from the lowered kinetic and thermal constraints; however, the identification of a cold shock−induced ribosome-binding protein factor that inhibits translation suggests that a more active process may be operating². In E. coli, this protein factor is encoded by the gene yfiA, and termed RaiA (ribosome-associated inhibitor) or protein Y (PY)^{5, 6}. The solution structures of PY^{7, 8} as well as of a PY homolog from Haemophilus influenzae⁹ reveal the protein to have a globular domain comprising two -helices and four -sheets, followed by a flexible C-terminal tail. PY has been shown to bind to small (30S) ribosomal subunits and 70S ribosomes, which are stabilized against dissociation, but no binding to large (50S) ribosomal subunits has been detected⁵. During cold shock, or upon entry into stationary phase, PY associates with ribosomes^{6, 10}, where it has been proposed to interfere with elongation by decreasing the accuracy of translation and preventing the binding of aminoacyl-tRNAs to the A site of the ribosome⁶. However, the exact mechanism by which PY regulates translation and protects the ribosomes during cold shock has remained unclear.

Cate and co-workers¹ have addressed this issue using a combined structural and functional approach: the relatively small size of PY (<15 kDa) allowed the complete protein to be soaked into preformed crystals of 70S E. coli ribosomes. The structure of the resulting complex was solved at a resolution of 11 Å, and the known PY structure could be unequivocally fitted into the PY density. The binding site of PY is exclusively on the 30S subunit of the 70S ribosome, where it substantially overlaps with the known binding positions of the anticodon stems of A- and P-site tRNAs (Fig. 1). Footprinting and binding assays complemented this localization by showing competition between PY and tRNA binding at both A and P sites of the ribosome. PY protects the universally conserved bases G926 and C1400, which are diagnostic bases for the presence of a bound anticodon region of a P-tRNA¹¹, whereas the enhanced reactivity of A1493, an A-site residue involved in decoding¹², indicates that PY chases tRNA from the A site. PY inhibited binding of deacylated tRNA to the P site and was slightly more effective at the cold-shock temperature of 16 °C as compared with 37 °C. This trend was even more striking when the more physiological situation of initiator fMet-tRNA binding to the P site of programmed ribosomes was measured. At 37 °C, little effect on P-site occupation was observed in the presence or absence of PY, whereas at 16 °C, PY markedly reduced binding, especially in the absence of initiation factors (IFs). The stimulatory effect of IFs on fMet-tRNA binding is consistent with their in vivo role to promote binding of the initiator fMet-tRNA to the ribosome during initiation of protein synthesis. However, it should be noted that the initiation pathway generally progresses through a preinitiation complex involving only the 30S subunit, initiation factors and the fMet-tRNA. Because PY interacts exclusively with components of the 30S subunit, this suggests that a similar interplay among PY, IFs and fMet-tRNA would occur on the small subunit, although this remains to be confirmed. With respect to IF1, the partial overlap in binding site with that of PY could also contribute to the antagonistic effects that IFs have on PY activity.

Figure 1. The binding site of protein Y on the ribosome overlaps with A- and P-site tRNA. The relative position of protein Y (red) to A- (green), P- (blue) and E-site (orange) tRNA is shown on the background of a schematic representation of a 70S ribosome, composed of 30S and 50S subunits as indicated. Full Figure and legend (37K)

Figure 2. Scheme for protein Y−mediated regulation under cold-shock conditions. Under normal growth conditions (top panel, shaded red), run-off 70S ribosomes from translation are dissociated by the combined action of IF1 (blue) and IF3 (purple). In the presence of IF2, the ternary complex containing the initiator tRNA, IF2−GTP−fMet-tRNA, is delivered to the ribosome. Subunit joining leads to dissociation of the initiation factors, leaving the initiator tRNA at the P site (Pi complex). A decrease in temperature produces a cold shock (blue arrow) as seen in the bottom panel (shaded blue), which leads to upregulation in expression of cold-shock proteins such as protein Y (PY, red). PY binds to run-off 70S ribosomes at a position overlapping the A and P sites (see Fig. 1), preventing the binding of the initiation factors IF1 and IF3 and therefore the dissociation of the 70S ribosomes into its subunits. PY also prevents binding of mRNA and tRNA to the 70S ribosome, thus preventing translation initiation. When temperatures return to normal, PY has a lower affinity for the ribosome than tRNA and initiation factors, allowing ribosomes to follow the alternative pathway into active translation (red arrows). Full Figure and legend (57K)

Several other stress proteins in E. coli have been shown to interact with the ribosomes of stationary phase cells^{10, 13, 14, 15}. One of these, RMF (ribosome modulation factor), has been suggested to induce dimerization of 70S ribosomes^{13, 14}, although the inactivation of the ribosomes may be due to probable binding of this factor within the active site for peptide bond formation on the large subunit¹⁵, rather than the actual dimerization itself. Another factor, termed YhbH, although having 40% identity to PY, was found to be associated exclusively with the dimerized 70S ribosomes, rather than 70S monosomes, suggesting that it may have a function distinct to PY during the stress response. Homologs for PY have also been identified in chloroplasts and in some cyanobacteria. The PY homolog in spinach was found to be present on chloroplast ribosomes and was termed ribosomal protein S22, then later reassigned as a plastid-specific ribosomal protein 1 (psrp1)¹⁶. The mature spinach chloroplast psrp1, when overexpressed in E. coli, was found to incorporate into 70S ribosomes and 30S subunits, but not into actively translating polysomes¹⁷. This may suggest that psrp1 is the chloroplast homolog of PY and not a ribosomal protein at all. However, it should be noted that the chloroplast and cyanobacterium PY homologs have considerably longer C-terminal extensions than PY and therefore may fulfill additional roles to that of PY. In this respect it is notable that a PY homolog in Synechococcus PCC 7002 termed lrtA (light repressed transcript) has been shown, as its name suggests, to be repressed by light—in other words, it is present under dark stress conditions where the protein synthesis machinery is less active¹⁸. Thus, the work of Vila-Sanjurjo et al.¹ adds further weight to the suggestion that PY may operate in several bacteria, chloroplasts and cyanobacteria under a variety of different stress conditions.

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