Nucleotide-sequence-specific and non-specific interactions of T4 DNA polymerase with its own mRNA.
The DNA-binding DNA polymerase (gp43) of phage T4 is also an RNA-binding protein that represses translation of its own mRNA. Previous studies implicated two segments of the untranslated 5'-leader of the mRNA in repressor binding, an RNA hairpin structure and the adjacent RNA to the 3' side, which contains the Shine-Dalgarno sequence. Here, we show by in vitro gp43-RNA binding assays that both translated and untranslated segments of the mRNA contribute to the high affinity of gp43 to its mRNA target (translational operator), but that a Shine-Dalgarno sequence is not required for specificity. Nucleotide sequence specificity appears to reside solely in the operator's hairpin structure, which lies outside the putative ribosome-binding site of the mRNA. In the operator region external to the hairpin, RNA length rather than sequence is the important determinant of the high binding affinity to the protein. Two aspects of the RNA hairpin determine specificity, restricted arrangement of purine relative to pyrimidine residues and an invariant 5'-AC-3' in the unpaired (loop) segment of the RNA structure. We propose a generalized structure for the hairpin that encompasses these features and discuss possible relationships between RNA binding determinants of gp43 and DNA binding by this replication enzyme.
Pavlov
A R
AR
Department of Biochemistry, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA.
Karam
J D
JD
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D004259:DNA-Directed DNA Polymerase; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic / Q000235:genetics; D017354:Point Mutation; D011485:Protein Binding; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D004259:DNA-Directed DNA Polymerase