Herpes simplex virus type 1 ribonucleotide reductase large subunit: regions of the protein essential for subunit interaction and dimerization.
We have constructed a series of random N-terminal deletions of the large subunit (R1) of the herpes simplex virus type 1 ribonucleotide reductase. Deletions extended throughout the R1 gene open reading frame and, in total, 31 different truncated polypeptides were expressed in Escherichia coli using the T7 expression system. N-Terminal truncations were analyzed for their interaction with the small subunit (R2) of ribonucleotide reductase using a sensitive enzyme-linked immunosorbent assay (ELISA) method and for their ability to complement R2 in ribonucleotide reductase assays. Truncated proteins were also tested for homodimerization using gel-filtration chromatography. The results identified a region of R1 between amino acids 349 and 373 which was essential for subunit interaction. Proteins lacking up to 348 amino-terminal residues associated with R2 and complemented R2 in ribonucleotide reductase assays. Proteins commencing at amino acid 373 and beyond did not interact with R2 and were inactive in enzyme assays. Using a plasmid which expressed an N-terminal deleted protein commencing at amino acid 247, we constructed two defined C-terminal deletions to give proteins comprising amino acids 247-434 and 247-996 of R1. Neither of these truncated proteins bound R2 and we concluded that a second region between amino acids 996 and 1137 (the C-terminus) is required for interaction with R2. Gel-filtration studies indicated that deletion of the first 420 amino acids from R1 did not affect dimerization. However, deletions of 457 amino acids and larger gave proteins which existed as monomers.(ABSTRACT TRUNCATED AT 250 WORDS)
Conner
J
J
Medical Research Council Virology Unit, Institute of Virology, Glasgow, United Kingdom.
Furlong
J
J
Murray
J
J
Meighan
M
M
Cross
A
A
Marsden
H
H
Clements
J B
JB
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002850:Chromatography, Gel; D004262:DNA Restriction Enzymes; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011994:Recombinant Proteins; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012264:Ribonucleotide Reductases; D004262:DNA Restriction Enzymes