Romeo T, Preiss J
Department of Biochemistry, Michigan State University, East Lansing 48824.
J Bacteriol. 1989 May;171(5):2773-82. doi: 10.1128/jb.171.5.2773-2782.1989.
Glycogen accumulation in Escherichia coli is inversely related to the growth rate and occurs most actively when cells enter the stationary phase. The levels of the three biosynthetic enzymes undergo corresponding changes under these conditions, suggesting that genetic control of enzyme biosynthesis may account for at least part of the regulation (J. Preiss, Annu. Rev. Microbiol. 38:419-458, 1984). We have begun to explore the molecular basis of this control by identifying factors which affect the expression of the glycogen genes and by determining the 5'-flanking regions required to mediate the regulatory effects. The in vitro coupled transcription-translation of two of the biosynthetic genes, glgC (ADPglucose pyrophosphorylase) and glgA (glycogen synthase), was enhanced up to 26- and 10-fold, respectively, by cyclic AMP (cAMP) and cAMP receptor protein (CRP). Guanosine 5'-diphosphate 3'-diphosphate stimulated the expression of these genes 3.6- and 1.8-fold, respectively. The expression of glgB (glycogen branching enzyme) was affected weakly or negligibly by the above-mentioned compounds. Assays which measured the in vitro formation of the first dipeptide of glgC showed that a restriction fragment which contained 0.5 kilobases of DNA upstream from the initiation codon supported cAMP-CRP-activated expression. Sequence-specific binding of cAMP-CRP to a 243-base-pair restriction fragment from the region upstream from glgC was observed by virtue of the altered electrophoretic mobility of the bound DNA. S1 nuclease protection analysis identified 5' termini of four in vivo transcripts within 0.5 kilobases of the glgC coding region. The relative concentrations of transcripts were higher in the early stationary phase than in the exponential phase. Two mutants which overproduced the biosynthesis enzymes accumulated elevated levels of specific transcripts. The 5' termini of three of the transcripts were mapped to a high resolution. Their upstream sequences showed weak similarity to the E. coli consensus promoter. These results suggest complex transcriptional regulation of the glycogen biosynthesis genes involving multiple promoter sites and direct control of gene expression by at least two global regulatory systems.
糖原在大肠杆菌中的积累与生长速率呈负相关，且在细胞进入稳定期时最为活跃。在这些条件下，三种生物合成酶的水平会发生相应变化，这表明酶生物合成的遗传控制可能至少部分解释了这种调节作用（J. 普赖斯，《微生物学年评》38:419 - 458，1984年）。我们已开始通过鉴定影响糖原基因表达的因素以及确定介导调节作用所需的5'侧翼区域，来探索这种控制的分子基础。环腺苷酸（cAMP）和环腺苷酸受体蛋白（CRP）分别将两种生物合成基因glgC（ADP葡萄糖焦磷酸化酶）和glgA（糖原合酶）在体外的偶联转录 - 翻译增强了26倍和10倍。鸟苷5'-二磷酸3'-二磷酸分别将这些基因的表达刺激了3.6倍和1.8倍。上述化合物对glgB（糖原分支酶）的表达影响微弱或可忽略不计。测量glgC第一个二肽体外形成的实验表明，一个包含起始密码子上游0.5千碱基DNA的限制性片段支持cAMP - CRP激活的表达。通过结合DNA电泳迁移率的改变，观察到cAMP - CRP与glgC上游区域一个243碱基对的限制性片段发生序列特异性结合。S1核酸酶保护分析确定了glgC编码区域0.5千碱基内四个体内转录本的5'末端。转录本的相对浓度在稳定期早期高于指数期。两个过量产生生物合成酶的突变体积累了更高水平的特定转录本。其中三个转录本的5'末端被高精度定位。它们的上游序列与大肠杆菌共有启动子有微弱的相似性。这些结果表明糖原生物合成基因存在复杂的转录调控，涉及多个启动子位点以及至少两个全局调节系统对基因表达的直接控制。
