The role of RbfA in 16S rRNA processing and cell growth at low temperature in Escherichia coli.
RbfA, a 30S ribosome-binding factor, is a multicopy suppressor of a cold-sensitive C23U mutation of the 16S rRNA and is required for efficient processing of the 16S rRNA. At 37 degrees C, DeltarbfA cells show accumulation of ribosomal subunits and 16S rRNA precursor with a significantly reduced polysome profile in comparison with wild-type cells. RbfA is also a cold-shock protein essential for Escherichia coli cells to adapt to low temperature. In this study, we examined its association with the ribosome and its role in 16S rRNA processing and ribosome profiles at low temperature. In wild-type cells, following cold shock at 15 degrees C, the amount of free RbfA remained largely stable, while that of its 30S subunit-associated form became several times greater than that at 37 degrees C and a larger fraction of total 30S subunits was detected to be RbfA-containing. In DeltarbfA cells, the pre-16S rRNA amount increased after cold shock with a concomitant reduction of the mature 16S rRNA amount and the formation of polysomes was further reduced. A closer examination revealed that 30S ribosomal subunits of DeltarbfA cells at low temperature contained primarily pre-16S rRNA and little mature 16S rRNA. Our results indicate that the cold sensitivity of DeltarbfA cells is directly related to their lack of translation initiation-capable 30S subunits containing mature 16S rRNA at low temperature. Importantly, when the C-terminal 25 residue sequence was deleted, the resulting RbfADelta25 lost the abilities to stably associate with the 30S subunit and to suppress the dominant-negative, cold-sensitive phenotype of the C23U mutation in 16S rRNA but was able to suppress the 16S rRNA processing defect and the cold-sensitive phenotype of the DeltarbfA cells, suggesting that RbfA may interact with the 30S ribosome at more than one site or function in more than one fashion in assisting the 16S rRNA maturation at low temperature.
Xia
Bing
B
Department of Biochemistry, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854-5635, USA.
Ke
Haiping
H
Shinde
Ujwal
U
Inouye
Masayori
M
D002455:Cell Division / Q000502:physiology; D003080:Cold Temperature; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D046911:Macromolecular Substances; D009154:Mutation; D012323:RNA Processing, Post-Transcriptional; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D012336:RNA, Ribosomal, 16S; C092748:RbfA protein, E coli; D012269:Ribosomal Proteins; D014357:Trypsin