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内容記述 |
In prokaryotes, the control of transcription initiation is a<br /> key step in the regulation of gene expression. In order to<br /> reveal the mechanism how the order of transcription is<br /> determined among thousands of genes in a cell, it is important<br /> to understand the intrinsic promoter strength for individual<br /> genes (the term "promoter strength" refers to the relative rate<br /> of synthesis of full length RNA product from a given promoter).<br /> The level of transcription initiation is basically determined by<br /> the sequence of the promoter, the start signal of RNA synthesis.<br /> DNA sequence analyses of a wide variety of prokaryotic promoters<br /> have indicated that promoters for the major form of <i>Escherichia</i><br /> <i>coli</i> RNA polymerase (Eσ70) are composed of two conserved<br /> hexanucleotide sequences, TATAAT and TTGACA, which are located<br /> at 10 and 35 base-pairs, respectively, upstream of the<br /> transcription initiation site, although a considerable variation<br /> exists in the promoter sequence between genes within the same<br /> organism. From thermodynamic and kinetic studies, these two<br /> sequences are believed to determine the affinity to RNA<br /> polymerase and the rate of DNA opening, altogether affecting the<br /> promoter strength. However, little is known about the role of<br /> individual bases within these two regions with respect to RNA<br /> polymerase binding and DNA opening. In this study, I carried out<br /> a systematic analysis of the relationship between the promoter<br /> sequence and the promoter strength (Kobayashi, M. <i>et al.</i> (1990)<br /> Nucleic Acids Res., <b>18,</b> 7367-7372).<br /> A set of 18 variant ,lacUV5 promoters was constructed, each<br /> carrying a single base substitution within the promoter -35<br /> region (nucleotide positions from -36 to -31 relative to the<br /> transcription start site). Using truncated DNA fragments<br /> carrying these variant promoters and purified Escherichia coli<br /> RNA polymerase holoenzyme(Eσ7O), the in vitro mixed<br/> transcription assays were performed to determine two parameters<br /> governing the promoter strength, <i>i. e.,</i> the binding affinity to<br /> RNA polymerase (parameter I) and the rate of open complex<br /> formation (parameter II).<br /> Parameter I was affected to various extents, while parameter<br /> II was mostly decreased except for two variant promoters, 34G<br /> and 33G (the variant promoters were named according to the<br /> position and base species of substitution). The 34G has a<br /> sequence of TTGACA, which is completely identical with the<br /> consensus sequence. The degree of change in parameter I mainly<br /> depends on the position of base substitution. Base substitutions<br /> at position -31 gave only a little effect; substitutions of C at<br /> position -32 to any other base caused significant reduction;<br /> base substitutions at position -35 also led to reduction,<br /> although the effects were somewhat smaller than those of -32<br /> base substitutions; the effects of base substitutions at<br /> position -33, -34 and -36 were variable depending on the base<br /> introduced. Among all possible sequences, TTGACA should be the<br /> strongest promoter in terms of parameter I. The rate of open<br /> complex formation (parameter II) was slower for most variant<br /> promoters than for the reference promoter, except for the 34G<br /> (consensus) and the 33G promoters. Again the promoter with the<br /> consensus TTGACA sequence was the strongest.<br /> In order to confirm these results, I next performed an<br /> abortive initiation assay, in which the formation of initial<br /> oligonucleotides is measured. The reaction conditions of the<br /> abortive initiation assay were made identical to those of the<br /> mixed transcription assay, except that ApA was added as a<br /> primer, and ATP, GTP and CTP were omitted (and thus [α-32P]UTP<br /> was a sole substrate). The final level indicates the binding<br /> affinity to RNA polymerase (parameter I'), while the reciprocal<br /> of the time required for reaching plateau level represents the<br /> rate of open complex formation (parameter II'). The pattern of<br /> the promoter strength determined by the abortive initiation<br /> assay was essentially the same as that for the mixed<br /> transcription assay. The degree of change in parameter I' is due<br /> to both the position and species of base substitution. However,<br /> all variant promoters except for 34G, displayed lower values of<br /> parameter II' than the reference promoter. In the case of<br /> parameter II', TTGACA was the only exception that was stronger<br /> than the reference promoter, but all other base substitutions<br /> resulted in marked reduction to less than half the level of the<br /> reference promoter. The alteration pattern of both parameter I'<br /> and II', measured by the abortive initiation assay, was<br /> essentially identical with that of parameter I and II determined<br /> by the in vitro mixed transcription assay.<br /> As an attempt to compare the promoter strength of the<br /> synthetic promoters measured by two in vitro assays with <i>in vivo</i><br /> activities, I performed β-galactosidase assay using variant<br /> lacUV5 promoter collections fused to the lacZ structural gene.<br /> The DNA fragments containing variant lacUV5 promoters were<br /> inserted between the inducible ara promoter and the lacZ,<br /> structural gene of plasmid vector pMS4342. I examined six<br /> variant promoters, which all showed unique promoter strength<br /> patterns in vitro. The promoter strength <i>in vivo</i> was determined<br /> simply by monitoring β-galactosidase activity in the absence of<br /> arabinose. The promoter 34G was as strong as the reference, and<br /> the promoters 33G and 31T were intermediate while the others<br /> were weak (less than 25%) . When compared with the results of two<br /> in vitro transcription assays, the promoter strength <i>in vivo</i> is<br /> in good agreement with parameter I measured by the productive<br /> initiation assay. The consensus sequence (34G) again exhibited<br /> the highest activity.<br /> The following conclusions were drawn from the data presented:<br /> (1) Alteration in the promoter strength of variant promoters is<br /> dependent on both the position and base species of<br /> substitutions; (2) the consensus sequence (TTGACA) exhibits the<br /> highest values for both parameters; (3) base substitutions at<br /> nucleotide position -34 cause marked effect on both parameters;<br /> (4) cytosine at nucleotide position -32 cannot be replaced with<br /> other nucleotides without significant reduction of the promoter<br /> strength; (5) base substitution at nucleotide position -31<br /> exerts only a little effect on parameter I; (6) the promoter<br /> strength in vivo is in good agreement in parameter I of <i>in vitro</i><br /> promoter strength; and (7) the consensus sequence (TTGACA)<br /> exhibits the highest activity <i>in vivo</i> as well as <i>in vitro.</i><br /> This type of experiments has been ｄone as a collaboration<br /> research for the analysis of sequence-strength relationship of<br /> the promoter -10 region. |
要旨・審査要旨 / Abstract, Screening Result (349.9 kB)
