Rehman, Majib Ur; (1995) Analysis of the transfer region of the Streptomyces plasmid SCP2*. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

pIJ903 is a bifunctional derivative of the 31.4kb low copy number, conjugative Streptomyces coelicolor plasmid SCP2*. pIJ903 was used in order to determine the complete nucleotide sequence of a critical part (3.35kb) of the transfer region (Sazcl (23) - PstI (18)). Three complete putative open reading frames with the codon usage typical of Streptomyces genes have been identified: traA (479 amino acids [aa]), traB (138 aa) and traX (291aa). The deduced sequences of none of the putative proteins showed any overall similarity to known protein sequences. traA is essential for SCP2* intermycelial plasmid transfer and pock formation and it encodes a protein of molecular weight 50,389 Daltons. The TraA protein contains a nucleotide binding sequence and shows a faint similarity to DNA helicases, suggesting that it, like Tra of pIJl0l and TraSA of pSAM2 might be a DNA helicase. traB encodes a putative protein of MW 14,703 Daltons with a potential leucine zipper motif, suggesting that it may be a DNA binding protein. traX would encode a protein of 30,587 Daltons but no function can be attributed to this protein as yet. The traB gene has been cloned into pT7-7 creating pQR524, a construct which produces a 16,100 Dalton fusion protein in vivo in E.coli. This TraB fusion protein has been used as an antigen in order to raise polyclonal rabbit antibodies against it. The TraB antibodies were shown to bind to TraB in E.coli but no TraB was detected in Streptomyces crude protein extracts. The DNA sequence proposed in this work is slightly different to that previously published. The divergence in the sequences causes a shift of the translational start codon of traA, such that traA would be translated into a protein 36 amino acids longer at its N-terminus. Other than the difference at the N-terminus the TraA proteins are very similar. Sequence analysis of the traX and traA indicated that these two genes may be translationally coupled, with the stop codon of traX overlapping the start codon of traA. In vitro protein analysis of the traX and traA genes in E.coli suggests that these genes synthesise a protein of approximately 80,000 Daltons (the sum of TraX and TraA). Further analysis suggests that the 80,000 Dalton protein produced in vitro could be a fusion protein, maybe the result of ribosomal frameshifting.

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