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聚合酶的转录启动需要噬菌体编码的转录激活子MoTA蛋白和T4共激活子Asia蛋白。这个系统是一个简单的模型，用来研究各种因素如何改变聚合酶的特异性。T4中间启动子包含一个由RNA聚合酶的sigma-70亚基识别的-10区域和一个以-30为中心的Mota盒序列，该序列由Mota结合。以前，我们已经确定了MoTA与MoTA盒结合的表观解离常数为130 nm。我们通过研究MoTA盒序列5TTTGCTTTA3(中间启动子的-34到-26位)中碱基决定因素的丢失或修改如何影响MoTA功能来扩展这项研究。将-30位的C：G碱基改变为A：T、G：C或T：A导致了一个与MOTA结合的MoTA盒，并且在体外对MoTA/Asia依赖的转录具有活性。胸腺嘧啶-29位上的5-甲基残基有助于MoTA结合，使表观解离常数降低4倍。相反，将T：A AT-32转换为C：I BP，这一变化影响主要凹槽，但不影响次要凹槽，产生了与野生型相似的MoTA盒。此外，即使将-34到-32位置的T：A比特替换为C：I比特，也只是适度地损害了结合。综上所述，这些结果表明，MOTA在中心GC的上游使用小槽触点，在中心GC的下游使用主要沟槽触点，在位置-30的C：G BP处不需要任何特定的基础特征。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都不能抑制MOTA和聚合酶/MOTA/ASIA与中间启动子PuvsX的结合，这表明-30位上游的小槽结合不需要与任何特定的T：A碱基接触。ASIA是一种由90个氨基酸组成的蛋白质，与RNA聚合酶的sigma-70亚单位紧密结合。ASIA抑制宿主启动子的转录，并在MoTA存在的情况下共同激活T4中间启动子的转录。亚洲的C末端含有异常多的疏水氨基酸，其中苯丙氨酸位于第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，因此我们将亚洲的C-末端区域作为突变目标。我们构建了一个亚洲突变体，其中第81和83个氨基酸的酪氨酸被改变为丙氨酸(ARA)，亚洲突变体的C端缺失4个氨基酸、12个氨基酸或17个氨基酸。野生型Asia在大肠杆菌中表达时具有极强的毒性，可能是因为它与sigma-70紧密结合。缺失4个和12个氨基酸的Asia蛋白和ARA蛋白在大肠杆菌中表达时也是有毒的。这些突变的亚洲蛋白还补充了T4亚洲琥珀突变噬菌体在非抑制条件下的生长。相反，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，使用纯化的蛋白，我们发现每个突变的Asia蛋白都与sigma-70结合，并在体外发挥辅助激活作用。我们的结果表明，虽然在体外纯化的系统中不需要ASIA的C末端17个氨基酸来发挥功能，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、激活

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