Effects of chaperones on mRNA stability and gene expression in Escherichia coli

Effects of chaperones on mRNA stability and gene expression were studied in order to develop an efficient Escherichia coli expression system that can maximize gene expression. The stability of mRNA was modulated by introducing various secondary structures at the 5'-end of mRNA. Four vector systems providing different 5'-end structures were constructed, and genes encoding GFPuv and endoxylanase were cloned into the four vector systems. Primer extension assay revealed different mRNA half-lives depending on the 5'-end secondary structures of mRNA. In addition to the stem-loop structure at the 5'-end of mRNA, coexpression of dnaK-dnaJ-grpE or groEL-groES, representative heat-shock genes in E. coli, increased the mRNA stability and the level of gene expression further, even though the degree of stabilization was varied. Our work suggests that some of the heat-shock proteins can function as mRNA stabilizers as well as protein chaperones.     

Coupling between H+ entry and ATP formation in Escherichia coli.

Chiral α-amino acid esters react readily with fluorescamine (Fluram^®) to form pyrrolinone-type chromophores. The characteristic Cotton effects given by these chromophoric derivatives provide a means for the determination of the absolute configuration of the parent amino acid ester. The strong CD band in the 330-320 nm region of the L-amino acid ester derivative is always negative, while it is positive for the D-amino acid ester derivatives. A pyrrolinone chirality rule is proposed to explain the sign of this Cotton effect.     

Coupling between codon usage, translation and protein export in Escherichia coli

Proteins destined for export via the Sec-dependent pathway are synthesized with a short N-terminal signal peptide. A requirement for export is that the proteins are in a translocationally competent state. This is a loosely folded state that allows the protein to pass through the SecYEG apparatus and pass into the periplasm. In order to maintain pre-secretory proteins in an export-competent state, there are many factors that slow the folding of the pre-secretory protein in the cytoplasm. These include cytoplasmic chaperones, such as SecB, and the signal recognition particle, which bind the pre-secretory protein and direct it to the cytoplasmic membrane for export. Recently, evidence has been published that non-optimal codons in the signal sequence are important for a time-critical early event to allow the correct folding of pre-secretory proteins. This review details the recent developments in folding of the signal peptide and the pre-secretory protein.     

Interactions between mutations affecting ribosome synthesis in Escherichia coli

RNA synthesis was followed during amino acid starvation of strains of Escherichia coli that contained both the relaxed (relA) mutation and a mutation affecting ribosome assembly that results in oversynthesis of RNA. The ribosome mutation did not by itself lead to relaxedness. The relaxed mutation could be expressed in organisms that contained the ribosome mutation.     

Characterization of a specific interaction between Escherichia coli thymidylate synthase and Escherichia coli thymidylate synthase mRNA.

Previous studies have shown that human TS mRNA translation is controlled by a negative autoregulatory mechanism. In this study, an RNA electrophoretic gel mobility shift assay confirmed a direct interaction between Escherichia coli (E.coli) TS protein and its own E.coli TS mRNA. Two cis-acting sequences in the E.coli TS mRNA protein-coding region were identified, with one site corresponding to nucleotides 207-460 and the second site corresponding to nucleotides 461-807. Each of these mRNA sequences bind TS with a relative affinity similar to that of the full-length E.coli TS mRNA sequence (IC50 = 1 nM). A third binding site was identified, corresponding to nucleotides 808-1015, although its relative affinity for TS (IC50 = 5.1 nM) was lower than that of the other two cis-acting elements. E.coli TS proteins with mutations in amino acids located within the nucleotide-binding region retained the ability to bind RNA while proteins with mutations at either the nucleotide active site cysteine (C146S) or at amino acids located within the folate-binding region were unable to bind TS mRNA. These studies suggest that the regions on E.coli TS defined by the folate-binding site and/or critical cysteine sulfhydryl groups may represent important RNA binding domains. Further evidence is presented which demonstrates that the direct interaction with TS results in in vitro repression of E.coli TS mRNA translation.     

Degradation of mRNA in Escherichia coli

Degradation of messenger RNAs (mRNAs) is a universal process that occurs in every cell and has important implications for nucleotide metabolism and gene expression. One organism in which mRNA degradation has been thoroughly studied is the bacterium Escherichia coli (E. coli). In this review I describe what is presently known about the different processes involved in the conversion of mRNAs from high molecular weight species to mononucleotides in E. coli. The ribonucleases and accessory factors involved in mRNA degradation, and features on mRNAs that make them resistant or sensitive to degradation will also be described. At the conclusion of this review, some of the anticipated directions of future research on this topic will be discussed.     

大肠杆菌异源表达的indigoidine与靛蓝的稳定性比较

【背景】Indigoidine是一种来源于微生物的无毒天然蓝色素。【目的】比较Indigoidine和靛蓝的色素稳定性,进而评价Indigoidine的色素稳定性。【方法】构建重组菌株Escherichia coli DH5α/p28s异源表达Indigoidine,考察可见光、紫外线、pH、温度、氧化还原剂、食品添加剂和金属离子对其与商品级靛蓝色素稳定性的影响。【结果】以N,N-二甲基甲酰胺为溶剂,Indigoidine和靛蓝都对可见光、紫外线敏感;2种色素在pH1.0-11.0时稳定,强碱性pH对色素破坏作用很大;Indigoidine抗Vc还原能力强于靛蓝,氧化剂可不同程度地降低2种色素的保存率;2种色素热稳定性不佳,在75℃以下时Indigoidine的色素稳定性优于靛蓝;食品添加剂中的柠檬酸和苯甲酸分别对Indigoidine和靛蓝均具有显著的护色效果;对这2种色素,Ca²⁺、Mg²⁺均具有护色效果,Na⁺、K⁺、Li⁺总体上没有明显的破坏作用,而Zn²⁺、Al³⁺、Cu²⁺、Fe²⁺、Fe³⁺则具有显著的破坏作用。【结论】Indigoidine色素稳定性明显优于靛蓝,具有广阔的开发应用前景。     

Analysis of mRNA synthesis following induction of the Escherichia coli SOS system

Escherichia coli responds to impairment of DNA synthesis by inducing a system of DNA repair known as the SOS response. Specific genes are derepressed through proteolytic cleavage of their repressor, the lexA gene product. Cleavage in vivo requires functional RecA protein in a role not yet understood. We used mRNA hybridization techniques to follow the rapid changes that occur with induction in cells with mutations in the recA operator or in the repressor cleavage site. These mutations allowed us to uncouple the induction of RecA protein synthesis from its role in inducing the other SOS functions. Following induction with ultraviolet light, we observed increased rates of mRNA synthesis from five SOS genes within five minutes, maximum expression ten to 20 minutes later and then a later decline to near the initial rates. The presence of a recA operator mutation did not significantly influence these kinetics, whereas induction was fully blocked by an additional mutation in the repressor cleavage site. These experiments are consistent with activation of RecA protein preceding repressor cleavage and derepression of SOS genes. The results also suggest that the timing and extent of induction of individual SOS genes may be different.