Protein determinants of RNA binding by DNA polymerase of the T4-related bacteriophage RB69.
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.
Petrov
Vasiliy M
VM
Department of Biochemistry, Tulane University Health Sciences Center, New Orleans, Louisiana 70112, USA.
Ng
San-San
SS
Karam
Jim D
JD
D000409:Alanine / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D008751:Methylene Blue / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
D003432:Cross-Linking Reagents; D006023:Glycoproteins; D012333:RNA, Messenger; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D012313:RNA; D004247:DNA; D017868:Cyclic AMP-Dependent Protein Kinases; D004259:DNA-Directed DNA Polymerase; C463461:bacteriophage RB69 DNA polymerase; D003546:Cysteine Endopeptidases; C019913:clostripain; D000409:Alanine; D008751:Methylene Blue