Erwinia herbicola Eho13類胡蘿蔔素操縱子的起動子分析

Abstract

Erwinia herbicola Eho 13屬於腸內菌科(Enterobacteriaceae)，是一種會生成黃色素的細菌。前人的研究發現此黃色素即是類胡蘿蔔素(carotenoid)。在這段carotenoid生合成基因中，共包含五個基因（依序為crt EXYIB）。本論文針對此基因群進行起動子區域的DNA序列分析，並決定起動子的位置。針對此基因群中具起動活性的EcoRI-EcoRV及PstI-Hind Ⅲ片段進行DNA序列分析，分別得到423 bp及1235 bp的序列。Primer extension及基因融合的研究發現在423 bp中具有一正向的起動子(crt P1)及一反向起動子(crt P2)，二者皆位於此基因群的第一個基因(crtE)的上游。crt P1的轉錄起始點位於423 bp中的nucleotide 42, crt P2的轉錄起始點位於423 bp中的nucleotide 66-68左右，二者所轉錄出來的RNA有25-27 bp的互補序列。在1235 bp中則定位出另一crt mRNA transcript的5'端，即位於nucleotide 145；但以RCR增殖出nucleotide 43-178的DNA片段進行基因融合卻沒有任何表現起動子活性的選殖體，表示在nucleotide 43-178中很可能並無起動子存在，或其須要位於其下游的crtB'（比crtB少28個氨基酸序列，與crtB具有共同的C-terminal end序列）基因產物的調節。PCR的研究發現相鄰基因間皆有mRNA transcript相連，推測此基因群很可能形成一操縱子。然而，根據RNA slot blot的分析結果，發現Tn1000的插入（其插入位置位於crtP1下游的nontranslated leader region）造成crtE(ORF-A)的轉錄表現降低，但crtI(ORF-D)的表現卻未因此而下降，表示在crtB(ORF-E)上游可能具有內在起動子。因此，Eho 13 carotenoid類胡蘿蔔素基因群具有相當複雜的構造，推測其除了共同由crtP1所起動外，還包含了內在起動子。其反向起動子的存在可能扮演基因調控的角色，而其結構之證明及內在起動子之定位將有助於crt基因之表現及調節機制的瞭解。

Erwinia herbicola Eho13 is a Gram-negative bacterium, belonging to the family of Enterobacteriaceae. This organism is yellow-pigmented. Previous studies showed that the pigments synthesized by this organism are carotenoids. The genes encoding carotenoid biosynthesis in E. herhicola Ehol3 has been cloned. These genes are clustered in a 6365-bp fragment which consists of 5 genes, crtEXYIB. The main goal of this thesis was to determine the location of the crt promoters. Previous gene-fusion studies showed that two promoters were located in the EcoRI-EcoRV and Pst I-Hind Ⅲ fragments. Sequencing results revealed that the EcoRI-EcoRV fragment was 423-bp long and the PstI-HindⅢ fragment was 1235-bp long. Primer extension studies have identified three 5' RNA termini in the 423-bp fragment at position of n.t. 42, 66-68, and 164. Gene fusion studies revealed that two of these three RNA species were transcribed from two different promoters. These two promoters (crtP1 and crtP2) were transcribed from an opposite direction and the transcripts, from position 42 and from 66-68, overlapped approximately 25-27 bp. A transcription-start site (n.t. 145) was also located in the 1235-bp fragment by primer extension. However, gene fusion studies with a DNA fragment containing n.t. 43-n.t. 178 failed to show promoter activity. These results suggested that this promoter, if present, may be regulated by a regulator that was absent in the gene-fusion study. If this promoter (crtP3) was functional in vivo, the sequence may encode a protein that was 28-amino acids shorter at the N-terminal end than the protein encoded by the last gene of the gene cluster, crtB. RNA PCR results suggested that the carotenoid-synthesis genes are expressed as an operon because of the connections of crt mRNA transcripts between the genes. However, there are possible internal promoters located at the upstream sequence of crtB (ORF-E) according to the results of RNA slot blot analysis. Therefore, the structure of Eho13 carotenoid-synthesis genes are complex, and the promoter crt P2 may play a regulatory role in the gene cluster. The demonstration of ne structure of crt operon and the locations of the internal promoters will be an essentiaisteep toward the understanding of the expression and the regulation of crt genes.