Terminal Proteins

Archetypal TPs

The first Streptomyces TPs characterized were found to be highly conserved in size (184-185 amino acids (aa)) and sequence (Fig. 2a) (Bao and Cohen 2001; Yang et al. 2002). No similarity to other known protein sequences in the databases (including the TPs of adenoviruses and ^29) was found. They are herein designated archetypal TPs to distinguish them from the heterologous TP that caps SCP1 (see below). Bao and Cohen designated the gene encoding archetypal TPs as tpg followed by a letter (and sometimes a number) to identify its origin. For example, the three TP genes for the linear plas-mids pSLA2-L, pSLA2-M, and chromosome in S. rochei were named tpgRl, tpgR2, and tpgR3, respectively (Bao and Cohen 2001; Mochizuki et al. 2001; original assignments of Bao and Cohen corrected based on unpublished results communicated by H. Kinashi), and the TP genes for the S. coelicolor and S. lividans chromosomes were designated tpgC and tpgL. This genetic nomenclature is unconventional and is proving confusing as the catalog grows. A more conventional use of only "tpg" (gene) and "Tpg" (gene product) for generic designation is therefore used here. TP (terminal protein) is a general term applicable (as it has been elsewhere) to all those proteins that cap the DNA of linear plasmids and chromosomes, including those of adenoviruses, ^29 phage, and any other such replicons. In Streptomyces, TPs include Tpgs as well as others of heterologous origins (e.g., TP of SCP1—see below). To specify the origin of a tpg gene or Tpg protein, the plasmid name or a three-letter abbreviation of the species (as for restriction enzyme nomenclature) will be added in superscript. For example, the Tpgs of SLP2 and the S. coelicolor chromosome are designated TpgSLP2 and TpgSco, respectively.

The amino acid sequences of the Tpg homologs contain a putative helix-turn-helix domain that shows limited similarity to the DNA-binding "thumb" domain of HIV reverse transcriptase (Bao and Cohen 2001; Yang et al. 2002) and putative amphiphilic ^-sheets (Fig. 2a). The latter may participate in protein-protein or protein-membrane interactions, as observed in aden-oviruses and ^29 phage (Ortin et al. 1971; Schaack et al. 1990; Bravo and Salas 1997).

Most functional tpg form a polycistronic operon with an upstream gene tap, which is also essential for replication of the linear replicons in Streptomyces (Bao and Cohen 2003). In S. coelicolor and S. lividans, the tap-tpg operon is located about 100 and 120 kb, respectively, from the "right" end of the chromosome (based on the conventional orientation of the S. coelicolor chromosome). On the S. avermitilis chromosome, the tapSav-tpgSav operon lies about 8 kb from the right end—corresponding to the left end of the

Fig. 2 Structure of TPs. a Aligned archetypal Tpg sequences. For a plasmid-encoded Tpg, the plasmid name is given, and for a chromosome-encoded Tpg, the three-letter abbreviation of the species is given (Sav, S. avermitilis; Sco, S. coelicolor; Sli, S. lividans; Sro, S. rochei; Ssc, S. scabies). pFRLl encodes two Tpgs. These two Tpgs are distinguished by their gene numbers (6 and 9). The amino acid residues are shaded using the Rasmol color scheme. Four identified features (based on TpgSco and TpgSl1) are indicated. HIV RT, human immunodeficiency virus reverse transcriptase; HTH, helix-turn-helix DNA-binding domain; NLS, nuclear localization signal. b Tpc of SCP1

Fig. 2 Structure of TPs. a Aligned archetypal Tpg sequences. For a plasmid-encoded Tpg, the plasmid name is given, and for a chromosome-encoded Tpg, the three-letter abbreviation of the species is given (Sav, S. avermitilis; Sco, S. coelicolor; Sli, S. lividans; Sro, S. rochei; Ssc, S. scabies). pFRLl encodes two Tpgs. These two Tpgs are distinguished by their gene numbers (6 and 9). The amino acid residues are shaded using the Rasmol color scheme. Four identified features (based on TpgSco and TpgSl1) are indicated. HIV RT, human immunodeficiency virus reverse transcriptase; HTH, helix-turn-helix DNA-binding domain; NLS, nuclear localization signal. b Tpc of SCP1

S. coelicolor chromosome. Some linear Streptomyces plasmids also contain tpg homologs, which are sometimes pseudogenes. For example, SLP2 contains a unique tpgSLP2 gene on the left arm plus a tpg pseudogene on the 15.3-kb right arm, which is identical to the termini of the S. lividans chromosome. Smaller linear plasmids tend to lack a tpg homolog. They appear to be capped by the Tpgs encoded by larger plasmids or the host chromosome (Bao and Cohen 2001). Many tpg pseudogenes and at least one functional tpg gene (tpgSLP2; C.-C. Yang, personal communication) are not accompanied by a tap gene.

Pseudogenes of tpg appear at unusually high frequencies in Streptomyces genomes. Their aberrant (usually shortened) size and the lack of a typical Streptomyces codon preference hint at their degeneracy. The first tpg pseudogene discovered was in the 15.3-kb terminal sequence shared by both arms (in TIR) of the S. lividans chromosome and the right arm of SLP2. This tpg pseudogene (encoding 183 aa) is about 8 kb from the telomere, and has been proven to be defective based on its inability to support replication of a recombinant linear plasmid (C.-C. Yang, unpublished results).

Putative tpg pseudogenes were also found in the chromosome of S. aver-mitilis (Ikeda et al. 2003). Their amino acid sequence is more divergent than that of the pseudogene in S. lividans, although their lack of function has not been confirmed. The terminal location of the tpg pseudogenes suggests that they are remnants of previous exchanges with other linear plasmids or chromosomes. Pseudogenes in the genomes of free-living bacteria are relatively rare, presumably because of their rapid degeneration under evolutionary selection. The finding of the tpg pseudogenes supports the occurrence of relatively recent end exchange events. The wide distributions of tpg genes and pseudogenes probably reflect the high mobility and frequent intermolecular exchanges of linear plasmids.

Nonarchetypal TPs: The TP of SCP1

The nonarchetypal telomere sequence of SCP1 and the absence of a tpg homolog on it suggested that it may be capped by a different type of TP. Recently the TP that caps SCP1 was isolated and identified. It is encoded by a gene (SCP1.127, designated tpc) on SCP1 (Huang et al. 2007). The gene product (Tpc; 259 aa) is significantly longer than the archetypal TPs (Fig. 2b), and does not resemble any protein in the databases. It contains an HTH DNA-binding domain in the N-terminal region, followed by a so-called guanine nucleotide transfer domain. These motifs are presumably involved in anchoring to the telomeric DNA with the guanine nucleotide transfer domain providing specificity for the terminal guanidine residues at the 5' end. A gene tac (SCP1.125) upstream of tpc, encoding a hypothetical protein, is also essential for replication of SCP1. Therefore, the SCP1 telomeric DNA and tac-tpc set represent a heterologous system that has evolved convergently to form a novel Streptomyces telomere system.

Plasmids pRL1 and pRL2 also contain nonarchetypal telomeres, and therefore may be capped by novel TPs. pRL2 nevertheless encodes a Tpg homolog (TpgpRL2), which is significantly larger than archetypal Tpgs (216 vs 184-185 aa). TpgpRL2 could support replication of an artificially constructed linear plasmid with pRL2 telomeres in the absence of tpg, suggesting that it capped the pRL2 telomeres (Z. Qin, personal communication). However, it is not clear whether in vivo pRL2 is also capped by TpgpRL2 or by another TP. In contrast, pRL1 contains no homolog of tpg or tap.

TP-TP Interactions

Interaction between the TPs at the two telomeres was initially suggested based on the immobilization of the TP-DNA complex during electrophoresis without a denaturing or proteolytic treatment (for example, Lin et al. 1993). TP-TP interaction (between two TpgSco) at the telomeres in vivo was recently demonstrated by chemical cross-linking experiments and atomic force microscopy by Tsai et al. (unpublished results). If such a TP-TP interaction persists during recombination, an even number of crossovers would be required to resolve two recombining chromosomes, and this would automatically lead to circular genetic maps (Stahl and Steinberg 1964; Stahl 1967). Indeed, the genetic maps obtained from various Streptomyces species have always been circular (Hopwood 1967) despite the linearity of their chromosomes (Lin et al. 1993). This model was further supported by the observed strong bias toward an even number of crossovers during recombination of Streptomyces chromosomes (Wang et al. 1999). For a more comprehensive explanation, see the recent review by Hopwood (2006).

TP-TP interactions have previously been demonstrated in other TP-capped genomes, including ^29 phage (Ortin et al. 1971), GA-1 phage (Arnberg and Arwert 1976), and adenoviruses (Robinson et al. 1973). In the absence of proteolytic treatment, these linear genomes assume a circular form (Arnberg and Arwert 1976; Keegstra et al. 1977; Wong and Hsu 1989). The identical functions of these heterologous TPs in diverse biological systems (bacterial chromosomes, plasmids and phages, and animal viruses) indicate the importance of this function.

Association of telomeres in vivo makes sense in that it provides topo-logical constraints that make superhelicity in the linear DNA possible, which would be important for replication, transcription, and/or recombination. Indeed, superhelical structures of adenovirus DNA were detected in the virus particles (Wong and Hsu 1989). Persistent telomere interaction, on the other hand, creates a postreplicational segregation complication that is also generally faced by circular replicons (see below).

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