Segments of Escherichia coli genome similar to the exons of human prothymosin α gene

Abstract

Identtficauon of tile putatwe prothymosin ~ homolo8 in gscherichia colt cells prompted the search for a prothymosm or.coding gone in the E cob genome. A ~ct of interspersed DNA segments was ldenufied, which match various parts of the human prothymosm ~ molecule. Their location in the E coh genome and high degree of simdanty with the appropriate regions of the human prothymosin cc gone suggest that some kind of ttcma.sphcing should e~lst tn £. colt, which could be responsible for bringing these putative bacterial prothymosin a-coding cxons together.

Multiple tRNA attachment sites in prothymosin α

“…Production of carboxy‐terminal (His) 6 ‐tagged rat ProTα and its derivatives in E. coli cells was driven by pQE60 and pQE70 vectors (QIAGEN) carrying corresponding inserts of the protein encoding regions of rat ProTα cDNA. Construction of the parental pQN3 plasmid encoding the full‐length rat ProTα and the plasmid encoding ProTα mutant I was described previously [7]. For constructing a plasmid encoding ProTα mutant III, pQN3 was digested with Dde I, blunt‐ended with the Klenow fragment in the presence of dNTP and subsequently cleaved with Eco RI.…”

“…ProTα mutants and their complexes with tRNA were isolated from E. coli JM109 cells carrying appropriate plasmids as described for full‐length ProTα [7]. Procedures for radiolabelling of the ProTα‐linked tRNA and fractionation of the ProTα‐tRNA complexes and tRNA‐peptides by Ni‐NTA affinity chromatography and polyacrylamide gel electrophoresis (PAGE) were reported previously [7].…”

“…Another nuclear protein involved in proliferation of the mammalian cells, prothymosin α (ProTα), was reported to link a short RNA fragment of an unknown origin in mouse Krebs 2 cells [5,6]. Recently, the ability of ProTα to link an RNA was mimicked in Escherichia coli cells producing recombinant mammalian ProTα [7]. In this case, the RNA moiety of the complex was characterized and proved to contain a set of tRNAs, including tRNA Lys , tRNA Ser 3 , tRNA Ile 2 and tRNA Met m [7].…”

“…Recently, the ability of ProTα to link an RNA was mimicked in Escherichia coli cells producing recombinant mammalian ProTα [7]. In this case, the RNA moiety of the complex was characterized and proved to contain a set of tRNAs, including tRNA Lys , tRNA Ser 3 , tRNA Ile 2 and tRNA Met m [7]. As in mouse cells, the ProTα‐linked bacterial tRNAs possess free 3′ termini and 5′ termini that are blocked, evidently by ProTα.…”

“…As in mouse cells, the ProTα‐linked bacterial tRNAs possess free 3′ termini and 5′ termini that are blocked, evidently by ProTα. A tRNA attachment site was mapped in the carboxy‐terminal half of ProTα [7] where its nuclear localization signal resides [8], raising the possibility that tRNA linking could influence the subcellular localization of the protein. Here, by using a deletion mutagenesis analysis of ProTα, we present evidence that there are several tRNA linking sites within the ProTα molecule.…”