BACTERIOPHAGE T4 GENE EXPRESSION

正常的细胞发育需要RNA聚合酶与许多调节转录活性的因子相互作用。我们研究了噬菌体T4中间启动子的转录，其中大肠杆菌RNA聚合酶启动转录需要噬菌体编码的转录激活剂MotA蛋白和T4辅助激活剂AsiA蛋白。该系统是一个简单的模型，用于检查因素如何改变聚合酶的特异性。T4中间启动子包含一个被RNA聚合酶sigma-70亚基识别的-10区域和一个以-30为中心被MotA结合的MotA盒状序列。先前，我们已经确定了MotA与MotA盒子结合的表观解离常数为130 nM。我们通过研究MotA框序列5TTTGCTTTA3（中间启动子的- 34至-26位）中碱基决定子的缺失或修饰如何影响MotA功能来扩展这项研究。将-30位的C:G bp改变为A:T、G:C或T:A，产生一个MotA盒子，该盒子被MotA结合，并在体外对MotA/AsiA依赖性转录具有活性。胸腺嘧啶-29位的5-甲基残基是一个主要的凹槽接触，有助于MotA结合，使表观解离常数降低4倍。相比之下，将-32的T:A转换为C:I bp，这一变化影响了大调槽，但不影响小调槽，产生了与野生型相似的MotA框。此外，即使用C:I bps取代-34至-32位的T:A bps，也只会轻微损害结合。综上所述，这些结果表明，MotA在中心GC的上游使用了小槽接触，下游使用了大槽接触，并且在位置-30的C:G bp处不需要任何特定的碱基特征。然而，我们的甲基化干扰分析表明，鸟嘌呤和腺嘌呤的预甲基化既没有抑制MotA的结合，也没有抑制聚合酶/MotA/AsiA与中间启动子PuvsX的结合，这表明-30位上游的轻微凹槽结合不需要与任何特定的T:A bp接触。AsiA是一种由90个氨基酸组成的蛋白质，与RNA聚合酶的sigma-70亚基紧密结合。在MotA存在的情况下，AsiA抑制宿主启动子的转录，并作为T4中间启动子转录的共激活因子。亚洲的c端含有异常多的疏水氨基酸，苯丙氨酸位于第73和77位，酪氨酸位于第81、83和87位。由于参与蛋白质-蛋白质或蛋白质- dna相互作用的蛋白质结构域可能具有这种类型的氨基酸序列，因此我们针对亚洲的c端区域进行诱变。我们构建了亚洲突变体，其中氨基酸81和83的酪氨酸变成了丙氨酸（ARA），亚洲突变体的c端缺失了4 aa、12 aa和17 aa。野生型AsiA在大肠杆菌中表达时毒性极大，可能是因为它与sigma-70紧密结合。缺失4和12个氨基酸的AsiA蛋白和ARA蛋白在大肠杆菌中表达时也具有毒性。这些突变的亚洲蛋白也补充了T4亚洲琥珀突变噬菌体在非抑制条件下的生长。相比之下，缺失17个氨基酸的蛋白质毒性较低，并且不能补充突变噬菌体。然而，使用纯化蛋白，我们发现每个突变的AsiA蛋白都与sigma-70结合，并在体外作为共激活剂发挥作用。我们的研究结果表明，虽然在体外纯化系统中，AsiA的c -末端17氨基酸不是功能所必需的，但这些氨基酸可能有助于AsiA蛋白在体内的功能或稳定性。-转录，噬菌体，活化

Normal cell development requires the interaction of RNA polymerase with many factors that regulate transcriptional activity. We study transcription from bacteriophage T4 middle promoters in which the initiation of transcription by E. coli RNA polymerase requires the phage-encoded transcriptional activator, MotA protein, and the T4 co- activator, AsiA protein. This system is a simple model for examining how factors can change the specificity of a polymerase.T4 middle promoters contain a -10 region that is recognized by the sigma-70 subunit of RNA polymerase and a MotA box sequence centered at -30 that is bound by MotA. Previously, we have determined an apparent dissociation constant of 130 nM for MotA binding to a MotA box. We have extended this study by investigating how the loss or modification of base determinants within the MotA box sequence 5TTTGCTTTA3 (positions - 34 to -26 of a middle promoter) affects MotA function. Changing the C:G bp at position -30 to either an A:T, G:C, or T:A resulted in a MotA box that was bound by MotA and was active for MotA/AsiA dependent transcription in vitro. The 5-methyl residue on the thymine at position -29, a major groove contact, contributed to MotA binding, decreasing the apparent dissociation constant by a factor of 4. In contrast, converting the T:A at -32 to a C:I bp, a change that affects the major but not the minor groove, yielded a MotA box that was bound similarly to wild type. Furthermore, even replacing the T:A bps at positions -34 through -32 with C:I bps only modestly impaired binding. Taken together, these results suggest that MotA uses minor groove contacts upstream and major groove contacts downstream of the center GC and does not require any specific base feature at the C:G bp at position -30. However, our methylation interference analyses indicated that neither the binding of MotA nor the binding of polymerase/MotA/AsiA to the middle promoter PuvsX was inhibited by premethylation of guanines and adenines, suggesting that minor groove binding upstream of position -30 does not require contact with any specific T:A bp. AsiA is a protein of 90 amino acids that binds tightly to the sigma-70 subunit of RNA polymerase. AsiA inhibits transcription from host promoters and acts as a co-activator of transcription from T4 middle promoters in the presence of MotA. The C-terminal portion of AsiA contains an unusually high number of hydrophobic amino acids, with phenylalanines at positions 73 and 77 and tyrosines at positions 81, 83, and 87. Because protein domains that are involved in protein-protein or protein-DNA interactions can have this type of amino acid sequence, we targeted the C-terminal region of AsiA for mutagenesis. We constructed an AsiA mutant in which the tyrosines at amino acids 81 and 83 were changed to alanines (ARA) and AsiA mutants with C-terminal deletions of 4 aa, 12 aa, or 17 aa. Wild type AsiA is extremely toxic when expressed in E. coli, presumably because of its tight binding to sigma-70. The AsiA proteins with deletions of 4 and 12 amino acids and the ARA protein were also toxic when expressed in E. coli. These mutant AsiA proteins also complemented a T4 asiA amber mutant phage for growth under nonsuppressing conditions. In contrast, the protein with the deletion of 17 amino acids was less toxic, and it did not complement the mutant phage. However, using purified proteins, we found that each of the mutant AsiA proteins bound to sigma-70 and functioned as a co-activator in vitro. Our results suggest that while the C-terminal 17 amino acids of AsiA are not required for function in a purified in vitro system, these amino acids may contribute to the function or stability of the AsiA protein in vivo. - transcription, bacteriophage, activation