Protein Determinants of RNA Binding by DNA Polymerase of the T4-related Bacteriophage RB69

Abstract

DNA polymerase (gp43) of phage T4 plays two biological roles, one as an essential DNA binding replication enzyme and the other as an mRNA-specific autogenous translational repressor. Binding of T4 gp43 to its mRNA target (translational operator RNA) interferes with gp43-DNA interactions, but it is unclear how the protein determinants for binding DNA are affected by the dynamics of gp43-mRNA interactions. We have used RB69 gp43, a natural variant of the T4 enzyme whose crystal structure has been determined to identify protein sites that respond to the interaction with specific RNA. We used protein phosphorylation markers, photocross-linking studies, protease sensitivity assays, and mutational analyses to examine the effects of operator RNA on the enzyme's five structural domains (N, exo, palm, fingers, and thumb). Our studies suggest that this RNA affects gp43-DNA interactions through global effects on protein structure that occlude DNA-binding sites but leave the enzyme accessible to interactions with the sliding clamp (RB69 gp45) and possibly other polymerase accessory proteins. We discuss the possible biological significance of putative RNA-binding motifs in the N and palm domains of RB69 gp43.

Diversity of structure and function of DNA polymerase (gp43) of T4-related bacteriophages

“…4, we highlight gp43 sites (based on the RB69 gp43 structure, Fig. 1) that have been implicated in RNA binding in our previous studies [12]. Conceivably, specific RNA ligands bind to these sites and play a role in bringing gp43A and gp43B subunits together during assembly of the dimeric enzyme.…”

“…Together, these three crite ria may contribute to the evolution of a wide spectrum of RNA targets for gp43 without necessarily requiring a major divergence in protein structure. In experiments that mapped sites on RB69 gp43 that interact with RNA, we concluded that multiple contacts in four of the five domains of the protein are required, with most contacts involving amino acid residues or sequence motifs that are highly conserved among gp43 variants [12]. Thus, the conserved structural features of this pol B DNA poly merase appear to have a plasticity to use alternative con tacts with potential RNA targets that can accommodate evolutionary change in the target sequence without loss of the ability to maintain the protein-RNA interaction nec essary for translational repression.…”

“…The ribbon diagram in Fig. 1 shows RB69 gp43 in com plex with primer template DNA [12] and also highlights regions that have been implicated in mRNA binding [13]. We use this structure as a reference to discuss other molecular forms of gp43 that we have discovered among the T4 like phages.…”

“…A ribbon diagram of the structure of RB69 gp43 complexed with primer template DNA. The diagram is modified from Franklin et al [13] to highlight sites on the gp43 molecule (black dots) that have been implicated in binding the mRNA tar get for the protein [12]. The same structural framework has been observed for archaeal DNA polymerases belonging to the pol B family [9 11].…”

Genetic Insertions and Diversification of the PolB-Type DNA Polymerase (gp43) of T4-Related Phages

“…The identities of the gp43A and gp43B polypeptides produced by the cloned wild-type 44RR gene (Fig. 4a, lane 1) were confirmed by N-terminal sequencing of the respective protein bands eluted from the gels using previously described methods 44 (data not shown). These observations rule out the possibility that the IC-UTS element provides signals for a translational bypass, 45,46 which would have generated a singlechain gp43 (or a gp43A-gp43B fusion protein).…”