Characterization of RNA primer synthesis in the T4 bacteriophage in vitro DNA replication system

In the presence of any natural single-stranded DNA, the bacteriophage T4 gene 41 and gene 61 proteins catalyze the synthesis of a group of pentaribonucleotides. These RNA molecules are homogeneous in chain length, but heterogeneous in sequence. In the presence of the other five T4 replication proteins, these pentaribonucleotides are utilized with high efficiency as primers for de novo DNA chain initiations on the lagging strand in the T4 DNA replication process. By using a two-dimensional fractionation system plus a variety of enzymatic analyses, the pentaribonucleotides are found to have either a unique pppApC dinucleotide or a pppG as their starting residue, followed in both cases by many different sequences. However, when natural T4 DNA containing glucosylated 5-hydroxylmethyl cytosine is used as template, only the pppApC-initiated class of RNA primers is made. Alone, the gene 41 protein exhibits a DNA-dependent GTPase (and ATPase) activity, with nucleoside diphosphate and inorganic phosphate as products. By blocking this GTPase activity with the synthetic GTP analogue, GTP gamma S, we show that limited RNa primer synthesis can occur without this nucleotide hydrolysis by the 41 protein, but that prolonged RNA primer synthesis requires such hydrolysis. This result, viewed in the light of the observations presented in the accompanying paper, suggests that the gene 41 protein acts as a "mobile promoter," perhaps leaving the gene 61 protein to perform the actual polymerization and chain termination functions in the synthetic process.