title: The roles of tryptophans in primer synthesis by the DNA primase of bacteriophage T7.
abstract: DNA primases catalyze the synthesis of oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Prokaryotic primases consist of a zinc-binding domain (ZBD) necessary for recognition of a specific template sequence and a catalytic RNA polymerase domain. Interactions of both domains with the DNA template and ribonucleotides are required for primer synthesis. Five tryptophan residues are dispersed in the primase of bacteriophage T7: Trp-42 in the ZBD and Trp-69, -97, -147, and -255 in the RNA polymerase domain. Previous studies showed that replacement of Trp-42 with alanine in the ZBD decreases primer synthesis, whereas substitution of non-aromatic residues for Trp-69 impairs both primer synthesis and delivery. However, the roles of tryptophan at position 97, 147, or 255 remain elusive. To investigate the essential roles of these residues, we replaced each tryptophan with the structurally similar tyrosine and examined the effect of this subtle alteration on primer synthesis. The substitution at position 42, 97, or 147 reduced primer synthesis, whereas substitution at position 69 or 255 did not. The functions of the tryptophans were further examined at each step of primer synthesis. Alteration of residue 42 disturbed the conformation of the ZBD and resulted in partial loss of the zinc ion, impairing binding to the ssDNA template. Replacement of Trp-97 with tyrosine reduced the binding affinity to NTP and the catalysis step. The replacement of Trp-147 with tyrosine also impaired the catalytic step. Therefore, Trp-42 is important in maintaining the conformation of the ZBD for template binding; Trp-97 contributes to NTP binding and the catalysis step; and Trp-147 maintains the catalysis step.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Huidong
authors.initials: H
authors.lastname: Zhang
authors.forename: Seung-Joo
authors.initials: SJ
authors.lastname: Lee
authors.forename: Charles C
authors.initials: CC
authors.lastname: Richardson
mesh_terms: D000255:Adenosine Triphosphate / Q000235:genetics / Q000378:metabolism; D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D019915:DNA Primase / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000096:biosynthesis* / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D007700:Kinetics; D009154:Mutation; D009843:Oligoribonucleotides / Q000096:biosynthesis / Q000235:genetics; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D013698:Templates, Genetic; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics; D015032:Zinc / Q000378:metabolism
chemical_list: D017931:DNA Primers; D004277:DNA, Single-Stranded; D009843:Oligoribonucleotides; D014764:Viral Proteins; D014443:Tyrosine; D014364:Tryptophan; D000255:Adenosine Triphosphate; D019915:DNA Primase; D015032:Zinc; D000409:Alanine