Analysis Of The Function Of Era And Era-interacting Factor

Homologues of Era, have been identified in all bacterium sequenced to date, and in some eukaryotic organisms, including mice and humans, form a new G-protein subfamily. In prokaryotic organism, Era is a GTPase essential for bacterial viability. Era appears to have an important role in the regulation of the bacterial cell-cycle and the processing and maturation of ribosome rRNA. Mutants that are partially defective in Era GTPase activity or that are reduced in the synthesis of wild-type Era become arrested in the cell-cycle at the predivisional two-cell stage and the suppression of ribosome maturation and assembly. In mammalian, Era is expressed in many tissues. Overexpression of mutated Era or the reduction of Era results in the arrest of cell cycle. However, the exact fuction of Era is still not clear. Therefore, it is significance to further study the function of Era for underdstanding the survival mechanism of organism as well as the physiological function of this new G-protein subfamily. In order to elucidate the function of Era, Chen NC of our group screened the Escherichia coli Era-associated protein from phage expression library of Escherichia coli genomic DNA using Dig-labeled Era protein. In this study, we identified the physical and physiological interaction between Era and YggG, and investigated the expression pattern and regulation of yggG gene in bacteria, and the function of YggG in E. coli cells. Furthermore, we investigated the expression pattern of Era in various tumors and in glandular epithelium and squamous epithelium. We also analyzed the expression pattern of hEra in adenocarcinoma and esophagus squamous cell carcinoma. On the base of these studies, we analyzed the function of Era in cells.The main results are as follow:1. YggG is an Era-binding protein.Using Dig-labeled Era protein to screen a phage expression library of Escherichia coli genomic DNA, we found that the product of yggG binds to Era. In this study, GST pull-down and co-immunoprecipitation assays furtherly showed that Era and YggG possessed physical interactions both in vitro and in vivo. In addition, we found that the mutated Era (Era-1) was also interacted with YggG. These results indicate that Era GTPase activity do not influence the interaction between Era and YggG.2. The expression pattern and regulation of yggG.yggG gene encodes a 25-kDa protein, a putative zinc metalloprotease. The deduced acid sequence of YggG showed high degrees of similarity to some other heat shock proteins. However, the exact location and regulation of yggG was not established previously. In this study, we firstly analyzed the 5'-end of the yggG mRNA by 5'-RACE (5'-rapid amplification of cDNA ends). Examination of the DNA sequences upstream of nucleotide + 1, the mRNA start site, reveals the presence of DNA sequences partly homologous to the -35 (TTCACC versus TTCAGA) and -10 (TAGAAT versus TATAAT) consensus sequences of The E. coli promoter. A possible ribosome-binding site consensus sequence (AAGAG) is present before the translational start site of yggG.To analyze the transcriptional regulation, we constructed a prokaryotic molecule report vector system pPlacZ. We mapped the promoter of yggG by constructing a set of nested deletions of the predicted yggG promoter region. These deletion fragments were fused to a lacZ reporter gene, andβ-galactosidase activity was measured for strains carrying each construct, both untreated and in heat shock condition. The fragment containing the 39 bp upstream from the yggG transcriptional start site was highly active, while the fragment containing the -339/-40 upstream from the transcriptional site of yggG had no promoter activity. These results indicate that the promoter of yggG is localized in the sequence -39/-1. Further study showed that the promoter activity of the fragment containing the -105/-1 upstream from the transcriptional start site of yggG increased in heat shock condition, while the fragment containing the -39/-1 upstream from the transcriptional start site of yggG was not regulated by heat shock. These results suggest that the sequence -105/-40 was responsible for the heat shock-induced transcriptional up-regulation of yggG.To confirm that the sequence -39/780 is necessary for the expression of predicted YggG protein, we introduced clone vectors of ORF1 (nucleotides -339 to 780) and clone vectors of ORF3 (nucleotides -39 to 780) into wild-type E. coli cells, respectively. The YggG protein from cells carrying the pUC/ORF1 or pUC/ORF3 was much greater than that from cells carrying only the chromosomal copy of the gene. The sequence of ORF2 (nucleotides -105 to 780) is similar to the coding sequence of yggG predicted by Genbank previously. After introduction of pDH2/ORF2 into W3110 cells for over-expressing ORF2, anti-YggG immunoblotting detected two bands: one was the over-expressed ORF2; the other was similar to the YggG detected in the wild-type E. coli, confirming that the coding sequence of yggG (ORF2) predicted previously by Genbank contains both the regulatory region and the coding sequence of yggG. These results support that the sequence -39/780 containing the promoter and coding sequence of yggG gene is necessary for the expression of predicted YggG protein.Experimental results further revealed that YggG was associated with the cytoplasmic membrane.In this study, we analyzed the transcriptional start site, promoter and stress-associated regulation region of yggG. Our results indicated that yggG gene encodes a 25kDa membrane-associated protein, and confirmed that yggG can not encode YggG (294aa) that was predicted by GenBank previously. The possible product of yggG is YggG (252aa), YggG (243aa) or YggG (231aa). Overproduction of YggG (252aa) revealed significant growth-inhibitory effect. To avoid this artificial effect, we used YggG(231aa) as a method in the study of YggG function.3. Era-1 mutant protein up-regulats the expression of yggG to alleviate the growth-inhibitory effect of Era-1, which promotes the survival of Escherichia coli cells containing Era-1 mutant protein.Transcriptional activity assay showed the expression of yggG-lacZ fusion in era-1mutants (BSP750) was not reduced with the decrease of cell growth rate and remained same expression level as wild type. However, in the strain HT120, the expression of this fusion was reduced to approximately 13% of wild type. RT-PCR and Real-Time PCR assay showed that yggG mRNA increased remarkably in BSP750 cells and the Escherichia coli cells overproducing Era-1. These results indicate that the Era-1 mutant proteins caused some stress, which stimulate the expression of yggG, indirectly or directly.To study the physiological role of YggG in E. coli, a targeted deletion mutant was generated by a recombination system for chromosome engineering in Escherichia coli. TheΔyggG strain has no observable phenotype. It grows as well as the parental strain under normal growth condition and high temperature. The presence of Era-1 may interfere with the assembly of 30S subunit and causes. Whether did YggG involve in this stress response and play a role? We compared the growth rate of wild-type E. coli cells overproducing Era-1 andΔyggG cells overproducing Era-1. The results indicated that over-expression of Era-1 in theΔyggG strain revealed more strong growth-inhibitory effect than that in the wild-type strain.We further analyzed the effect of over-expressed YggG proteins on the growth rate of era-1 mutants. The BSP750 cells overproducing YggG exhibited the same growth rate as the BSP750 cells carrying the control plasmid. It is thought that YggG has been increased in era-1 mutants and play a role. However, if YggG could inhibit the involvement of Era-1 in the assembly of 30S subunit, over-expression of YggG might improve bacterial growth rate. We over-expressed Era-1 or both YggG and Era-1 proteins simultaneously inΔyggG cells and compared their effects on bacterial growth. The results showed that the over-expression of Era-1 in the cell resulted in a reduction of growth rate, while the cells that co-expressed YggG and Era-1 revealed a moderate increase in growth rate. There was no difference in the expressional level of Era-1 between these two cells. These results indicate that the over-expression of YggG can alleviate the growth-inhibitory effect of overproducing Era-1. These results suggest that the expression of YggG increases with the reduction of Era GTPase activity, and YggG might be involved in the physiological function of Era in cell. In addition, these results indicate that the mutation, Era, interferes with the assembly of 30S subunit and causes some translational stress, which induces some stress-response factors to express. YggG, a heat shock protein, play a role in this stress response.4. Analysis of the expression pattern and function of human Era.We investigated the individual disparity of human Era expression by immunohisto- chemistry. We investigated the expression pattern of hera in various tumor tissue by in situ hybridization. The results showed that the expression level of hEra is relative higher in digestive cancer and adenocarcinoma. We analyzed the expression of hEra in human clone adenocarcinoma tissues and corresponding para-cancerous normal tissues colleted from 40 specimens and in esophagus squamous cell carcinoma and corresponding para-cancerous normal tissues colleted from 40 specimens of clinical operation, and then data were analyzed by statistics software. The results showed that the expression of hEra is significantly higher in glandular epithelium than that in squamous epithelium (P<0.01). Compared with corresponding para-cancerous normal tissues, the expression level of hEra in clone cancer reduced significantly (P<0.01). The expression of hEra decreased with the reduction of differentiated level of adenocarcinomas. However, hEra expression has no significant difference between esophagus squamous cell carcinoma tissues and corresponding para-cancerous normal tissues. These results suggested that hEra might be involved in the development of adenocarcinomas.Together, our results indicated that YggG is associated with the function of Era. YggG might play a role in maintaining the homeostasis of cells in some stress caused by Era mutant proteins. In human, Era might be involved in the development of adenocarcinomas.