Raab R, Neal G, Garrett J, Grimaila R, Fusselman R, Young R
J Bacteriol. 1986 Sep;167(3):1035-42. doi: 10.1128/jb.167.3.1035-1042.1986.
A plasmid carrying the bacteriophage lambda lysis genes under lac control was subjected to hydroxylamine mutagenesis, and mutations eliminating the host lethality of the S gene were selected. DNA sequence analysis revealed 48 single-base mutations which resulted in alterations within the coding sequence of the S gene. Thirty-three different missense alleles were generated. Most of the missense changes clustered in the first two-thirds of the molecule from the N terminus. A simple model for the disposition of the S protein within the inner membrane can be derived from inspection of the primary sequence. In the first 60 residues, there are two distinct stretches of predominantly hydrophobic amino acids, each region having a net neutral charge and extending for at least 20 residues. These regions resemble canonical membrane-spanning domains. In the model, the two domains span the bilayer as a pair of net neutral charge helices, and the N-terminal 10 to 12 residues extend into the periplasm. The mutational pattern is largely consistent with the model. Charge changes within the putative imbedded regions render the protein nonfunctional. Loss of glycine residues at crucial reverse-turn domains which would be required to reorient the molecule to reenter the membrane also inactivate the molecule. Finally, a number of neutral and rather subtle mutations such as Ala to Val and Met to Ile are found, mostly within the putative spanning regions. Although no obvious explanation exists for this subtle and heterogeneous class of mutations, it is noted that all of the changes result in a loss of alpha-helical character as predicted by Chou-Fasman theoretical analysis. Alternative explanations for some of these changes are also possible, including a reduction in net translation rate due to substitution of a rare codon for a common one. The model and the pattern of mutations have implications for the probable oligomerization of the S protein at the time of endolysin release at the end of the vegetative growth period.
一个携带在乳糖控制下的噬菌体λ裂解基因的质粒经过羟胺诱变处理，并筛选出消除S基因宿主致死性的突变。DNA序列分析揭示了48个单碱基突变，这些突变导致S基因编码序列内的改变。产生了33个不同的错义等位基因。大多数错义变化集中在分子从N端开始的前三分之二区域。通过检查一级序列可以推导出一个关于S蛋白在内膜中分布的简单模型。在前60个残基中，有两个明显的主要由疏水氨基酸组成的片段，每个区域都有净中性电荷且延伸至少20个残基。这些区域类似于典型的跨膜结构域。在该模型中，这两个结构域作为一对净中性电荷螺旋跨越双层膜，N端的10到12个残基延伸到周质中。突变模式在很大程度上与该模型一致。假定嵌入区域内的电荷变化使蛋白质失去功能。关键反向转折结构域处甘氨酸残基的缺失（这是分子重新定向以重新进入膜所必需的）也使分子失活。最后，发现了许多中性且相当细微的突变，如丙氨酸到缬氨酸以及甲硫氨酸到异亮氨酸的突变，大多发生在假定的跨膜区域内。尽管对于这类细微且异质的突变不存在明显的解释，但值得注意的是，所有这些变化都导致了如周-法斯曼理论分析所预测的α-螺旋特性的丧失。对于其中一些变化也可能有其他解释，包括由于用稀有密码子替代常见密码子而导致净翻译速率降低。该模型和突变模式对营养生长期结束时内溶素释放时S蛋白可能的寡聚化有影响。
