Weird facets of ribosome synthesis in stressfully chilled bacteria

Lolita Piersimoni, Mara Giangrossi, Claudio O. Gualerzi and Cynthia L. Pon Laboratory of Genetics, Department of Biology MCA, University of Camerino, 62032 Camerino (MC), Italy We present data indicating that an imbalance of initiation factors/ribosome is generated by a cold-shock-induced decrease of the rate of assembly of ribosomal subunits accompanied by an increased expression of initiation factors. The rRNA synthesized after cold shock (a 37°C→10°C temperature-downshift), unlike that synthesized during exponential growth at 37°C, is transcribed mainly from the P2 promoters, which become activated by stress. Furthermore, our data indicate that both synthesis and maturation of rRNA are substantially retarded after temperature downshift. The amount of rRNA synthesized de novo during and after cold-adaptation represents a fairly large amount of total RNA. The fate of the rRNA synthesized during cold-shock is that of-ending up primarily in the active ribosomes present in the polysomes, while the majority of the rRNA whose synthesis had started immediately before or after the stress ultimately ends up in presumably inactive 70S monomers. With respect to ribosomal proteins (r-proteins), we show that after cold-shock five r-proteins (S2, S3, S5, S6 and S19) give rise to two distinct spots in 2D-gel electrophoresis. The appearance of new spots for proteins S2, S3, S5 and S6 is due to the lack of some post translational modifications as determined by mass spectrometry analysis. In addition, the data indicate the presence of post translational modifications so far unknown. We also demonstrate that cold-shock does not affect the stoichiometry of the r-proteins within the 30S ribosomal subunits. The analysis of non-ribosomal proteins associated with the peak of 30S ribosomal subunits (which probably contains precursors of the 50S subunits) becomes specifically enriched in a number of proteins which are or might be involved in assembly and/or maturation of the ribosomal particles and translation. For example, among the initiation factors, IF2 is the only cold-shock induced initiation factor whose level increases in association with the 30S ribosomal subunit and whose function in cold-shock is actually not known. These findings open new perspectives towards the identification of a cold-shock role for this and other proteins. Supported by a MIUR PRIN 2007 Grant to CL