Phage Ø29 Protein p6 Is in a Monomer−Dimer Equilibrium That Shifts to Higher Association States at the Millimolar Concentrations Found <i>in Vivo</i>

Abril, Ana M.; Salas, Margarita; Andreu, José Manuel; Hermoso, José Miguel; Rivas, Germán

doi:10.1021/bi970994e

Cited by 39 publications

(56 citation statements)

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“…The in vivo accumulated protein p17 was calculated to be 15,000 molecules at 30 min post-infection (29), which is almost 45 M intracellular protein p17 concentration taking into account the cell volume of 10 Ϫ15 liters determined (35). Our in vitro results at 70 M protein concentration demonstrate that protein p17 forms dimers, in agreement with the finding of dimers in vivo.…”

Section: Resultssupporting

confidence: 84%

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Bacteriophage Φ29 Early Protein p17

Crucitti

Abril

Salas

2003

Journal of Biological Chemistry

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Gene 17 of the Bacillus subtilis phage ⌽29 is expressed early after infection, and it has been shown to be required at the very beginning of phage replication under conditions of low but not high multiplicity of infection. It has been proposed that, at the beginning of the infection, protein p17 could be recruiting limiting amounts of initiation factors at the viral origins. Once the infection process is established and the replication proteins reach optimal concentration, protein p17 becomes dispensable. In this paper we focused, on the one hand, on the study of protein p17 dimerization and the role of a putative coiled-coil region. On the other hand, we focused on its interaction with the viral origin-binding protein p6. Based on our results we propose that protein p17 function is to optimize binding of protein p6 at the viral DNA ends, thus favoring the initiation of replication and negatively modulating its own transcription.Processes like DNA replication, transcription, compaction, or recombination occur in all organisms, and all of them have to be regulated and coordinated in order to obtain biological activity. Factors involved in these regulations are found in both eukaryotes and prokaryotes. In eukaryotes there are proteins like HMG-1 (renamed HMGB1) and HMG-2 that enhance the DNA binding of a variety of proteins (1-4). In prokaryotes, there are histone-like proteins that can function in global regulation, acting as transcriptional modulators in multiprotein complexes with DNA. For example, H-NS influences transcription of a number of genes involved in diverse biological processes (5-9). Another histone-like protein, HU, has been shown to stimulate the action of different DNA-binding proteins (10 -12), to repress promoters together with other proteins forming high order multiprotein complex (13), to participate in the initiation of DNA replication assisting other replication proteins (14), or to be involved in post-transcriptional regulation (15). Generally, regulation requires homo-and/or hetero-protein-protein interactions, and different domains involved in these associations have been described: coiled-coiled (16), the RING-B box coiled-coil, renamed recently TRIM (17), EHV1 motif (18), or the Eps15 homology domain (19).Bacillus subtilis phage ⌽29 has a linear double-stranded DNA with a terminal protein (TP) 1 covalently linked to the 5Ј ends. ⌽29 DNA replication starts by recognition of the origins of replication, i.e. the TP-containing DNA ends, by a TP/DNA polymerase heterodimer (20). Protein p6 is a viral nonspecific DNA-binding protein that is involved in different DNA processes forming a nucleoprotein complex that plays an essential role in the initiation of ⌽29 DNA replication activating the replication origins (21,22). This complex has been shown to cover most of the ⌽29 genome, suggesting that p6 may also have a structural role in organizing the genome (23). Also, it has been demonstrated that protein p6 is involved in regulating the viral transcription; binding of protein p6 to the right ...

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Section: Resultssupporting

confidence: 84%

“…At late times after ⌽29 infection, the number of copies of protein p6 in B. subtilis cells has been calculated to be enough to cover the entire viral DNA progeny (35). The self-association ability of protein p6 was studied by analytical ultracentrifugation at the in vivo protein p6 concentration.…”

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confidence: 99%

Bacteriophage Φ29 Early Protein p17

Crucitti

Abril

Salas

2003

Journal of Biological Chemistry

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“…Nevertheless, under conditions that favor binding of p6 to DNA (low salt and saturating protein concentrations), p6-nucleoprotein complexes are observed covering most of the 29 genome, suggesting that p6 may also have a structural role in organizing the genome (Gutié rrez et al 1994). In agreement with this hypothesis, p6 concentrations at late times of infection can be as high as 1 mM, an amount sufficient to saturate not only the 29 replication origins but also the entire intracellular viral DNA (Abril et al 1997).…”

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confidence: 69%

Functional interactions between a phage histone-like protein and a transcriptional factor in regulation of phi 29 early-late transcriptional switch

Elías‐Arnanz

Salas²

1999

Genes & Development

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Protein p6 is a nonspecific DNA-binding protein occurring in high abundance in phage 29-infected cells. Here, we demonstrate a novel role for this versatile histone-like protein: its involvement in regulating the viral switch between early and late transcription. p6 performs this role by exhibiting a reciprocal functional interaction with the regulatory protein p4, also phage encoded, which is required for repression of the early A2b and A2c promoters and activation of the late A3 promoter. On the one hand, p6 promotes p4-mediated repression of the A2b promoter and activation of the A3 promoter by enhancing binding of p4 to its recognition site at PA3; on the other, p4 promotes p6-mediated repression of the A2c promoter by favoring the formation of a stable p6-nucleoprotein complex that interferes with RNA polymerase binding to PA2c. We propose that the observed interplay between proteins p6 and p4 is based on their DNA architectural properties. The process of transcription, like other DNA transactions such as replication and recombination, occurs on an inherently stiff molecule (see Shore and Baldwin 1983;Wang and Giaever 1988). However, DNA must be at least locally flexible for the appropriate nucleoprotein complexes to assemble. This requirement is partially fulfilled by the participation of DNA-binding proteins that have the ability to remodel DNA (Nash 1990(Nash , 1996Grosschedl et al. 1994;Werner and Burley 1997). Such architectural factors are found in both eukaryotes and prokaryotes and include specific as well as nonspecific DNA-binding proteins.Nonspecific architectural factors presumably regulate transcription by introducing conformational changes that either hinder or assist DNA recognition by sequence-specific control proteins. In prokaryotes, there is substantial evidence that factors capable of altering general DNA flexibility such as the major histone-like proteins HU and H-NS can also function as transcriptional modulators. Thus, HU has been shown to stimulate lac repressor-operator binding and CAP binding to lac DNA (Flashner and Gralla 1988), to stabilize Mu repressor binding to the Mu early operator (Betermier et al. 1995), to aid in GalR-mediated repression of the gal operon (Aki and Adhya 1997), and to displace the LexA repressor from its DNA-binding sites (Preobrajenskaya et al. 1994). Likewise, H-NS has been reported to influence transcription of a number of genes involved in diverse biological processes (Falconi et al. 1993;Zuber et al. 1994;Atlung and Ingmer 1997;Williams and Rimsky 1997).In eukaryotes, the abundant nonhistone chromosomal protein HMG-1 (and the highly homologous protein HMG-2) also functions as an architectural element in the assembly of several transcriptional nucleoprotein complexes (for review, see Grosschedl et al. 1994;Bustin and Reeves 1996). Thus, HMG-1 and HMG-2 have been shown to enhance the sequence-specific DNA binding of a variety of proteins, such as steroid receptors (Oñ ate et al. 1994;Verrier et al. 1997;Boonyaratanakornkit et al. 1998); the POU domain-...

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“…Careful biophysical characterization has previously allowed the elucidation of isodemic self‐association behavior for several other proteins, including insulin (Jeffrey et al , 1976), FtsZ (Rivas et al , 2000), Phage ϕ29 protein p6 (Abril et al , 1997), chicken deoxy hemoglobin D (Rana & Riggs, 2011), human apolipoprotein C‐II (Yang et al , 2012), tubulin (Frigon & Timasheff, 1975), and for the assembly of heterochromatin protein 1 with nucleosomes in controlling heterochromatin spread (Canzio et al , 2013). Isodesmic self‐association may be a general mechanism for creating large, labile polymers that can disaggregate readily when required.…”

Section: Discussionmentioning

confidence: 99%

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

et al. 2016

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Membrane‐less organelles in cells are large, dynamic protein/protein or protein/RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher‐order assemblies influences the recruitment of the speckle‐type POZ protein (SPOP) to nuclear speckles. SPOP, a cullin‐3‐RING ubiquitin ligase (CRL3) substrate adaptor, self‐associates into higher‐order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild‐type SPOP localizes to liquid nuclear speckles, self‐association‐deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB‐mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration‐dependent populations of the resulting oligomeric species. Higher‐order oligomerization of SPOP stimulates CRL3SPOP ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher‐order protein self‐association may be a general mechanism to concentrate functional components in membrane‐less cellular bodies.

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Phage Ø29 Protein p6 Is in a Monomer−Dimer Equilibrium That Shifts to Higher Association States at the Millimolar Concentrations Found in Vivo

Cited by 39 publications

References 44 publications

Bacteriophage Φ29 Early Protein p17

Bacteriophage Φ29 Early Protein p17

Functional interactions between a phage histone-like protein and a transcriptional factor in regulation of phi 29 early-late transcriptional switch

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

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