Ribosome‐associated protein that inhibits translation at the aminoacyl‐tRNA binding stage

Abstract

We have recently isolated and characterized a novel protein associated with Escherichia coli ribosomes and named protein Y (pY).Here we show that the ribosomes from bacterial cells growing at a normal physiological temperature contain no pY, whereas a temperature downshift results in the appearance of the protein in ribosomes. The protein also appears in the ribosomes of those cells that reached the stationary phase of growth at a physiological temperature. Our experiments with cell-free translation systems demonstrate that the protein inhibits translation at the elongation stage by blocking the binding of aminoacyl-tRNA to the ribosomal A site. The function of the protein in adaptation of cells to environmental stress is discussed. INTRODUCTIONThe involvement of ribosomes in low temperature adaptation of bacteria was implied a long time ago . It was reported that the ribosomal fraction of the bacterial cell is responsible for the arrest of translation at low temperatures . Some antibiotics specifically affecting bacterial ribosomes were shown to mimic cold shock or heat shock response, and so ribosomes were claimed to be sensors of cold and heat shock in bacteria . More recently, a ribosome-binding factor, RbfA, was discovered that proved to be a cold shock protein . RbfA was shown to bind with 30S ribosomal subunit and suppress a cold-sensitive mutation in 16S ribosomal RNA . Another cold shock protein, CsdA, was also characterized as a ribosome-associated protein, and its capacity to unwind doublestranded RNA was observed . The major cold shock protein of Escherichia coli, CspA (Goldstein et al., 1990), was reported to be an RNA-binding protein and qualified as an RNA chaperone . A number of other cold shock proteins were discovered in bacteria . In no case, however, were the mechanisms and effects of cold shock protein on protein synthesis and cell growth clarified. RESULTS AND DISCUSSIONThe presence of the recently discovered ribosome-associated protein pY in ribosomes was analysed depending on the conditions of the E. coli culture growth. To our surprise, the protein was not found in the ribosomal fraction of cells grown at the temperature for physiological activity in E. coli (37°C). The protein appeared in the ribosomal fraction when the cultivated cells were chilled rapidly to 15°C followed by incubation at the same temperature, or cooled gradually to 4°C. Figure 1 presents the area of the two-dimensional electrophoresis slab where pY migrates among ribosomal proteins. It can be seen that the proteins extracted from ribosomes of cells grown under normal physiological conditions contain very little, if any, pY ( Figure 1A), whereas a significant amount of the protein is + Corresponding author. Tel/Fax: +7 095 924 0493; E-mail: spirin@vega.protres.ru D.E. Agafonov, V.A. Kolb & A.S. Spirin scientific reports detected in the ribosomal fraction from cells subjected to a temperature downshift prior to harvesting ( Figure 1B). The appearance pY in some of the preparations of ribosomes from cells grown at ...

PSRP1 Is Not a Ribosomal Protein, but a Ribosome-binding Factor That Is Recycled by the Ribosome-recycling Factor (RRF) and Elongation Factor G (EF-G)

“…Consistently, we show that PSRP1 and pY inhibit the binding of tRNA to 70S ribosomes (light green bars in Fig. 3c), as has been reported previously for pY (17,33). pY was much more effective than PSRP1, and the presence of mRNA appeared to increase the stability of the tRNA on the ribosome (Fig.…”

“…8h) (41). Protein Y-stabilized ribosomes have been shown to be translationally inactive (33), consistent with the mapping of pY and PSRP1 to the site overlapping the mRNA and A-and P-tRNA binding sites (Fig. 3b).…”

Single-molecule investigations of the stringent response machinery in living bacterial cells

“…It also lays the foundation for forthcoming studies with a more detailed analysis of ribosomal distributions during the exponential phase (55), stationary phase (56)(57)(58), and during cold shock (59).…”

Ribosome‐associated factor Y adopts a fold resembling a double‐stranded RNA binding domain scaffold

“…Protein Y, the product of yfiA gene, is a ribosome-associated protein present in E. coli and many other bacteria [1][2][3]. pY was earlier assigned to the r 54 modulation protein family based on the observation that mutations in the related downstream r 54 gene cause an increase in the level of expression from r 54 -dependent promoters [4].pY (also called ribosome associated inhibitor, RaiA) [2] was detected in the ribosome fraction during environmental stress, as a consequence of either low temperature [2] or excessive cell density [2,3]. Furthermore, pY binds to the small ribosomal subunit in an Mg 2+ -dependent manner and becomes less exposed to solvent upon association of the small and large ribosomal subunits.…”