Flamm E L, Weisberg R A
J Mol Biol. 1985 May 25;183(2):117-28. doi: 10.1016/0022-2836(85)90206-2.
We describe the isolation and sequencing of the hip gene of Escherichia coli and show that it encodes the beta subunit of integration host factor (IHF beta). In order to locate the coding region, we constructed a set of deletion mutants by exonucleolytic digestion of a fragment containing hip, determined which mutants were hip+ and which hip- by complementation, and then sequenced the ends of the critical deletions. The 5' end of the coding region was located precisely by comparing the deduced amino acid sequence to the actual N-terminal amino acid sequence of IHF. Our assignment of the coding region was further substantiated by the nucleotide sequences of a hip point mutant and of internal replacement mutations. We found a probable promoter for hip located about 85 base-pairs upstream from the initial AUG codon and about 75 base-pairs downstream from the 3' end of the neighboring gene, rpsA, and we constructed an IHF beta overproducer by fusing the coding sequences to the lambda pL promoter. A survey of known protein sequences revealed a close relationship between IHF beta and the type II prokaryotic DNA binding proteins (the "histone-like" proteins). This relationship is shared to a considerable extent by the other subunit of IHF, IHF alpha. A hip missense mutation that replaces a completely conserved glycine with aspartate has a null phenotype, suggesting that the conserved regions are functionally important.
我们描述了大肠杆菌hip基因的分离和测序,并表明它编码整合宿主因子(IHFβ)的β亚基。为了定位编码区,我们通过对包含hip的片段进行核酸外切酶消化构建了一组缺失突变体,通过互补确定哪些突变体是hip+,哪些是hip-,然后对关键缺失的末端进行测序。通过将推导的氨基酸序列与IHF的实际N端氨基酸序列进行比较,精确确定了编码区的5'端。hip点突变体和内部置换突变体的核苷酸序列进一步证实了我们对编码区的定位。我们发现hip的一个可能启动子位于起始AUG密码子上游约85个碱基对处,相邻基因rpsA的3'端下游约75个碱基对处,我们通过将编码序列与λpL启动子融合构建了一个IHFβ过量产生菌株。对已知蛋白质序列的调查揭示了IHFβ与II型原核DNA结合蛋白(“组蛋白样”蛋白)之间的密切关系。IHF的另一个亚基IHFα在很大程度上也具有这种关系。一个将完全保守的甘氨酸替换为天冬氨酸的hip错义突变具有无效表型,表明保守区域在功能上很重要。