Ribitol dehydrogenase messenger RNA from an enzyme superproducer strain of Klebsiella aerogenes. Purification, cell-free translation and studies in vitro and in vivo.
1. Ribitol dehydrogenase messenger RNA, from a strain of Klebsiella aerogenes that had been evolved to superproduce this enzyme, has been purified in a single step by labelling extracted polysomes with rabbit anti(ribitol dehydrogenase) and immunoprecipitating with sheep anti-(rabbit IgG). 2. The extracted mRNA is stable in a protein synthesis system in vitro and directs synthesis 35-40-times more efficiently than RNA from coliphages MS2 or Q beta, to give ribitol dehydrogenase as sole major product. 3. Its size distribution shows a major band of 1500 nucleotides plus fragments 400-1400 nucleotides, with only traces of size 2400-3000 nucleotides. Only the latter could encode both proteins of the operon: ribitol dehydrogenase and D-ribulokinase. 4. Ribitol dehydrogenase mRNA represents 24% of total mRNA in cells harvested just after a 'switch' point' in mid-exponential phase. About half of the polysomes containing this mRNA are unattached to DNA, whereas only 3% of other mRNAs are unattached to DNA. 5. This mRNA is not outstandingly stable in vivo, though there are indications that it may be more stable than average. Hence the high level of synthesis of ribitol dehydrogenase (up to 30% of total protein in an extract) seems to be due to very efficient transcription and translation from multiple copies of a constitutive rbtD gene.
D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000201:enzymology*; D008970:Molecular Weight; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000096:biosynthesis; D012333:RNA, Messenger / Q000302:isolation & purification*; D012255:Ribitol / Q000235:genetics; D012293:Rifampin / Q000494:pharmacology; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics*
D012329:RNA, Bacterial; D012333:RNA, Messenger; D012255:Ribitol; D013401:Sugar Alcohol Dehydrogenases; C022529:ribitol 2-dehydrogenase; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin