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内容記述 |
Transcription initiation has long been assumed to be a sequence composed of four steps: binding of RNA polymerase to a promoter, isomerization of the resulting binary complex accompanied by strand opening, iterative synthesis and release of abortive transcripts, and escape of the enzyme from the promoter. These steps are either chemical step essential to synthesize long RNA or explain a phenomenon that is observed for all RNA polymerases examined. Although additional steps could exist, the sequence has been considered as the complete mechanism, mainly because of the lack of evidence for further complications. In fact, the sequential pathway explained the results previously obtained in particular for the initiation at the T7A1 promoter.<br />However, the existence of branched reaction pathway was recently discovered in the initiation at the λPRAL, lacUV5 and malT promoters, where a part of the enzyme-promoter complex is arrested at the promoter. This finding raises a new question which mechanism is more general and which is an exceptional case. From kinetic viewpoint, the sequential pathway is a special case of the branched pathway proposed. In a test of the generality of the pathway, one of the key criteria is whether or not initiation at the T7A1 promoter actually follows the branched pathway. I addressed this question by using several kinetic and biochemical techniques.<br />In the most sensitive kinetic assay, moribund complex, which synthesize only abortive transcripts as branched reaction, was detected and its amount increased in a low salt condition. In the gel mobility-shift assay and DNA footprinting with exonuclease III, a small amount of the complex arrested at the promoter was detected and its level was significantly raised in the low salt condition. The results of the DNA footprinting as well as that by Fe2+ cheleted at the active site of the enzyme demonstrated the existence of a fraction of binary complex that dislocates from its position of productive subspecies, suggesting that moribund subspecies is forward tracked compared with productive subspecies. This also suggests the existence of a branching point in the stage of binary complex that has been proposed for the initiation at the λPRAL. These results prove that the initiation at the T7A1 promoter also follows the common branched pathway.<br />The amount of arrested complex formed at the T7A1 promoter is less than 3% of that of binary complex that initially exist before adding NTPs. This explains the reason why the behaviors of the initiation at the promoter apparently follow the sequential pathway. The small fraction could be due to the rapid conversion between moribund and productive subspecies. The rapid conversion was confirmed by measuring the rates of their dissociation and the rates were 7 times faster than those for the λPRAL promoter. In conclusion, the branched pathway is more general mechanism and the case of T7A1 promoter is a particular one due to the rapid conversion.<br />I next addressed the question whether or not the branched pathway has physiological significance in E. coli cell. The clue is the effect of the transcription factors GreA and GreB on promoter arrest, which was previously shown to relieve the arrest in the initiation at the λPRAL promoter by introducing reversibility in the conversion of moribund subspecies into productive one.<br />An E.coli strain with disrupted greA and greB grows well in LB medium at 37 ℃, but does not grow at 25 ℃. It was also sensitive to Mn2+ or Zn2+-in LB medium. By using E.coli gene array, the candidate genes whose expressions are changed in the strain with disrupted greA and greB was previously selected. By using Northerm blotting, I confirmed that mRNA levels of at least 3 genes, cspA, rpsA and atpC, are decreased by the disruption of greA and greB. Full-length transcription from their promoters was observed to be enhanced in the presence of GreA or GreB in a purified reconstitution system. The addition of the factors reduced abortive transcripts, suggesting that the factors increased the yield of full-length transcript by mitigating the promoter arrest. Among the promoters tested, atp promoter, the major promoter of atp operon, was examined most detail. Primer extension analysis confirmed that the transcript transcribed from the atp promoter was indeed reduced by the disruption of greA and greB. The formation of moribund complex at the atp promoter was confirmed by the most sensitive kinetic assay. In conclusion, the branched pathway is working not only in vitro but also in E.coli, and the GreA and GreB regulate transcription of various genes by the mechanism. This finding also claims that the Gre factors are bonafide initiation factors, although they were discovered as elongation factors that mitigate elongation arrest in vitro. |
要旨・審査要旨 / Abstract, Screening Result (318.8 kB)
