‘Drc’, a structurally novel ssDNA-binding transcription regulator of N4-related bacterial viruses

Abstract

Bacterial viruses encode a vast number of ORFan genes that lack similarity to any other known proteins. Here, we present a 2.20 Å crystal structure of N4-related Pseudomonas virus LUZ7 ORFan gp14, and elucidate its function. We demonstrate that gp14, termed here as Drc (ssDNA-binding RNA Polymerase Cofactor), preferentially binds single-stranded DNA, yet contains a structural fold distinct from other ssDNA-binding proteins (SSBs). By comparison with other SSB folds and creation of truncation and amino acid substitution mutants, we provide the first evidence for the binding mechanism of this unique fold. From a biological perspective, Drc interacts with the phage-encoded RNA Polymerase complex (RNAPII), implying a functional role as an SSB required for the transition from early to middle gene transcription during phage infection. Similar to the coliphage N4 gp2 protein, Drc likely binds locally unwound middle promoters and recruits the phage RNA polymerase. However, unlike gp2, Drc does not seem to need an additional cofactor for promoter melting. A comparison among N4-related phage genera highlights the evolutionary diversity of SSB proteins in an otherwise conserved transcription regulation mechanism.

A Novel N4-Like Bacteriophage Isolated from a Wastewater Source in South India with Activity against Several Multidrug-Resistant Clinical Pseudomonas aeruginosa Isolates

“…A homolog of the LUZ7 Drc protein is present in the genome. Drc was recently elucidated as an SSB transcription factor that interacts with RNAP2, playing a role in the transition of early- to mid-phase gene transcription ( 17 ).…”

Unlimited cooperativity ofBetatectivirusSSB, a novel DNA binding protein related to an atypical group of SSBs from protein-primed replicating bacterial viruses

“…Nevertheless, dsDNA binding of B35SSB seems insignificant as only a very faint shifted band was observed (lane 12') at high a concentration of B35SSB (1000:1, B35SSB:dsDNA molar ratio), whereas B35SSB could shift nearly all ssDNA substrate at a 125:1 (B35SSB:ssDNA) molar ratio (lane 5). Recent works have also highlighted the RNA binding capacity of several SSBs from bacteria and viruses, leading to RNA metabolism regulation [24,36]. However, when RNA binding capacity of B35SSB was analyzed, only a single weak band was detected at a very high concentration of B35SSB (Figure 1B, lanes 7-12) indicating an inefficient binding to RNA, as compared with ssDNA of the equivalent sequence (Figure 1B, lanes 1-6).…”

“…This domain consists of a fivestranded β-sheet coiled to form a β-barrel capped by an α-helix [19][20][21]. There are very few exceptions of SSB structures with no clear similarity to an OB-fold, such as those in adenovirus [22], the hyperthermophilic archaea representing clade Thermoproteales [23] and Drc protein from the N4-related bacterial viruses [24].…”

Molecular Characterization and Comparative Genomic Analysis of vB_PaeP_YA3, a Novel Temperate Bacteriophage of Pseudomonas aeruginosa

“…It is well known that there are many unknown open reading frames termed 'ORFans' in phage genomes, especially in the early/delayed-early regions, and they are also less conserved between phages (Yin and Fischer, 2008). Several ORFans have been proven to mediate crucial processes such as host takeover and transcriptional regulation during phage infection (Roucourt and Lavigne, 2009;De Smet et al, 2017;Boon et al, 2019). Therefore, revealing the functions of ORFans in phage YA3 genome is believed to discover some distinct protein functions.…”