Cloning, Expression, Purification And Preliminary Crystallographic Analysis Of The EcTrmB And MjNGN

RNA molecules from all forms of life have been found to contain chemical modifications of nucleosides that are added to the RNA posttranscriptionally. These modifications are important for the correct function of RNAs and result in the production of mature RNAs. Among the different families of RNA molecules, tRNA is the most heavily modified. N7-Methylguanosine at position 46(m7G46) of tRNA is one of the most commonly modified nucleosides: it is widely found in eukaryotes and bacteria as well as some Archaea.The m7G46 modification is produced by a specific m7G46 tRNA methyltransferase. In eukaryotes, this enzyme is composed of two proteins subunits, Trm8p and Trm82p. The noncatalyic subunit Trm82p may regulate and help the catalytic subunit Trm8p to work. In contrast to eukaryotes, where the m7G46 methylation enzyme is a heterodimer, a single Trm8 subunit, named TrmB, is present in prokaryocyte. Both Trm8p and TrmB have the SAM binding domain. The m7G46 methylation enzyme belongs to the SAM-dependent methyltransferase family.So far, the crystal structures of bacterial TrmB proteins have been reported: those from Bacillus subtilis and Streptococcus pneumonia. Recently, the crystal structures of the yeast m7G46 methylation complex and its catalytic subunit Trm8p in complex with SAM have also been published. As the representative of monomeric TrmBs, the structure of Escherichia coli TrmB (EcTrmB) is still unknown. In order to understand the function and properties of TrmB proteins more clearly we attempts to crystallize full-length EcTrmB but failed. We designed three truncated protein lacking the first 18, 25, 32 residues of the N-terminus respectively. Finally, the truncated protein lacking the first 32 residues of the N-terminus but with an additional His6 tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P21.During all stages of transcription, the activity of RNA polymerase (RNAP) is highly regulated by numerous cis- or trans-acting elements. Transcription in archaea employs a eukaryotic-type transcription apparatus but uses bacterial-type transcription factors. NusG is one of the few archaeal transcription factors whose orthologs are essential in both bacteria and eukaryotes.Archaeal NusG is composed of only a NusG N-terminal (NGN) domain and a KOW domain, which is similar to bacterial NusG but not to the eukaryotic ortholog, Spt5. Spt5 proteins typically have an acidic N terminus, a single NGN domain, five or six KOW domains and a set of simple C-terminal repeats. Archaea represent one of the three domains of life, and are the closest relatives of the original cellular organisms. Archaeal NusG presents hybrid features of Spt5 and bacterial NusG. So, the research of Archaea NusG will help to understand the function and evolution of this essential transcription factor.We subcloned the coding sequence of the NGN domain from the synthesized Methanocaldococcus jannaschii NusG gene, and then expressed and purified the NGN protein. Then the NGN protein was crystallized by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P3221.