title: Mutational analysis of the terminal protein Tpg of Streptomyces chromosomes: identification of the deoxynucleotidylation site.
abstract: The linear chromosomes and linear plasmids of Streptomyces are capped by terminal proteins (TPs) covalently bound to the 5' ends of the DNA. The TPs serve as primers for DNA synthesis that patches in the single-stranded gaps at the telomeres resulting from the bi-directional replication ('end patching'). Typical Streptomyces TPs, designated Tpgs, are conserved in sequence and size (about 185 amino acids), and contain a predicted helix-turn-helix domain and a functional nuclear localization signal. The Tpg-encoding gene (tpg) is often accompanied by an upstream gene tap that encodes an essential telomere-associating protein. Five lone tpg variants (not accompanied by tap) from various Streptomyces species were tested, and three were found to be pseudogenes. The lone tpg variant on the SLP2 plasmid, although functional, still requires the presence of tap on the chromosome for end patching. Using a combination of in vitro deoxynucleotidylation, physical localization, and genetic analysis, we identified the threonine at position 114 (T114) in Tpg of Streptomyces lividans chromosome as the deoxynucleotidylated site. Interestingly, T114 could be substituted by a serine without destroying the priming activity of Tpg in vitro and in vivo. Such T114S substitution is seen in and a number of pseudogenes as well as functional Tpgs. T114 lies in a predicted coil flanked by two short helixes in a highly hydrophilic region. The location and structural arrangement of the deoxynucleotidylated site in Tpg is similar to those in the TPs of phage ø 29 and adenoviruses. However, these TPs are distinct in their sequences and sizes, indicating that they have evolved independently during evolution. Using naturally occurring and artificially created tpg variants, we further identified several amino acid residues in the N-terminus and the helix-turn-helix domain that were important for functionality.
authors: Department of Chemistry, Chung-Yuan Christian University, Chung-li, Taiwan. ccyang@cycu.edu.tw
authors: Chien-Chin
authors: CC
authors: Yang
authors: We-Chi
authors: WC
authors: Sun
authors: Wan-Yu
authors: WY
authors: Wang
authors: Chi-Hung
authors: CH
authors: Huang
authors: Fang-Shy
authors: FS
authors: Lu
authors: Shu-Min
authors: SM
authors: Tseng
authors: Carton W
authors: CW
authors: Chen
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D002876:Chromosomes, Bacterial / Q000235:genetics*; D004252:DNA Mutational Analysis; D004261:DNA Replication; D003854:Deoxyribonucleotides / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003854:Deoxyribonucleotides