Abstract
Escherichia coli cells lacking both polynucleotide phosphorylase (PNPase) and RNase PH, the only known P(i)-dependent exoribonucleases, were previously shown to grow slowly at 37 degrees C and to display a dramatically reduced level of tRNA(Tyr)su3+ suppressor activity. Here we show that the RNase PH-negative, PNP-negative double-mutant strain actually displays a reversible cold-sensitive phenotype and that tRNA biosynthesis is normal. In contrast, ribosome structure and function are severely affected, particularly at lower temperatures. At 31 degrees C, the amount of 50S subunit is dramatically reduced and 23S rRNA is degraded. Moreover, cells that had been incubated at 42 degrees C immediately cease growing and synthesizing protein upon a shift to 31 degrees C, suggesting that the ribosomes synthesized at the higher temperature are defective and unable to function at the lower temperature. These data indicate that RNase PH and PNPase play an essential role that affects ribosome metabolism and that this function cannot be taken over by any of the hydrolytic exoribonucleases present in the cell.
