Chin D T, Goff S A, Webster T, Smith T, Goldberg A L
Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 1988 Aug 25;263(24):11718-28.
To learn more about the mechanism and regulation of the ATP-dependent protease La in Escherichia coli, the lon gene was completely sequenced using the dideoxy method on fragments generated by Bal31 digestion. The predicted amino acid composition based on the DNA sequence agreed well with the composition of the acid-hydrolyzed protease. The predicted NH2-terminal amino acid sequence, tryptophan content, and the carboxyl terminus also agreed with experimental data. However, the molecular weight of 87,000 (783 amino acids) calculated from the DNA sequence was lower than prior estimates. The tetrameric enzyme contains four binding sites for ATP, a DNA-binding domain, a proteolytic site, and a regulatory site that binds unfolded polypeptides. An ATP-binding pocket exists on each subunit as shown by consensus sequences and elements of secondary structure resembling those on other nucleotide-binding proteins (e.g. adenylate kinase, RecA). For this purpose, improved consensus patterns for identifying ATP-binding domains were developed. Computer-assisted comparisons, however, failed to demonstrate any regions homologous to sequences in other polypeptides including proteases or DNA-binding proteins. This enzyme also contains an unusual highly acidic domain surrounded by very basic sequences. Protease La is the first ATP-dependent protease sequenced and seems to represent a new type of enzyme. The promoter sequence was similar to consensus sequences for other heat-shock promoters. Using site-directed mutagenesis, alterations were introduced into the putative promoter sequence. Mutations upstream of -35 had little effect, but alterations immediately upstream of -10 lowered basal transcription of a lon-lacZ operon fusion and reduced its response to inducers of the heat-shock response.
为了更深入了解大肠杆菌中依赖ATP的蛋白酶La的机制和调控，我们使用双脱氧法对经Bal31消化产生的片段进行测序，从而完整测定了lon基因的序列。基于DNA序列预测的氨基酸组成与酸水解蛋白酶的组成高度吻合。预测的氨基末端氨基酸序列、色氨酸含量以及羧基末端也与实验数据相符。然而，根据DNA序列计算得出的87,000（783个氨基酸）的分子量低于先前的估计值。该四聚体酶含有四个ATP结合位点、一个DNA结合结构域、一个蛋白水解位点以及一个结合未折叠多肽的调控位点。如共有序列和类似于其他核苷酸结合蛋白（如腺苷酸激酶、RecA）的二级结构元件所示，每个亚基上都存在一个ATP结合口袋。为此，我们开发了用于识别ATP结合结构域的改进共有模式。然而，计算机辅助比较未能证明与其他多肽（包括蛋白酶或DNA结合蛋白）的序列存在任何同源区域。这种酶还包含一个由非常碱性的序列包围的异常高酸性结构域。蛋白酶La是首个被测序的依赖ATP的蛋白酶，似乎代表了一种新型酶。其启动子序列与其他热休克启动子的共有序列相似。通过定点诱变，我们对推定的启动子序列进行了改变。-35上游的突变影响不大，但-10上游紧邻区域的改变降低了lon-lacZ操纵子融合体的基础转录水平，并减弱了其对热休克反应诱导剂的应答。
