Abstract
The amino acid sequence of purified gene 0.3 protein of T7, the protein responsible for overcoming host restriction, has been determined. The nucleotide sequence of the 0.3 RNA, the messenger RNA that codes for both the 0.3 protein and the gene 0.4 protein, a T7 protein of unknown function, has also been determined. The 0.3 RNA is 578 nucleotides long, 509 of which are used to code for the 2 proteins. The coding sequences do not overlap, but the termination codon for the 0.3 protein and the presumed initiation codon for the 0.4 protein do overlap in the sequence UAAUG. The 0.3 protein is very acidic: 34 of its 116 amino acids are aspartic or glutamic acid and only 6 are arginine or lysine. The 0.3 protein contains no cysteine. The nucleotide sequence predicts that the 0.4 protein consists of 50 amino acids and contains no histidine or proline. The effects of different mutations indicate that a protein which contains only the first 87 amino acids of the 0.3 protein is unable to prevent host restriction in vivo; one that contains te first 93 amino acids has weak function; and one that has the first 94 amino acids (plus 2 that are not in the wild type sequence) is fully able to prevent host restriction. The apparently critical 94th amino acid is tryptophan. The mutant 0.3 proteins that contain 87 or more amino acids appear to be reasonably stable in vivo, but those that contain 78 or fewer are apparently too unstable to have been observed by gel electrophoresis.
Highlights
The firstmessengerRNA made frombacteriophage T7 DNA during infection is the 0.3RNA (Studier et al, 1979b).’ The 0.3 RNA is released from longer primary transcripts by a single RNase I11 cleavage at each end (Dunn and Studier, 1973; Rosenberg and Kramer, 1977)
A table giving the restriction sites used for end-labeling of fragments and the extent of sequence determined from each site is given in the miniprintsection of this paper.? The changes caused by deletion and point mutations were found by isolating the appropriate restriction fragment from the DNA of purified mutant phage particles, determining thenucleotide sequence across the site of the mutation, and comparing the mutant and wild type sequences
The coding sequence for the 0.3 protein begins at theAUG initiation codon a t nucleotide 35 and ends at theUAA termination codon at nucleotide 386; the 0.4 protein apparently begins at theAUG at nucleotide 388 and ends at theUAG at nucleotide 541
Summary
Steitz and Bryan proposed that the0.4protein should begin at the AUG a t nucleotide 388 in the middle of the second protected fragment and utilize the sequence GAGGAGG (nucleotides 371-377) for initiation; asecond possible initiation site, the AUG at nucleotide 378 together with the sequenceGAGGAG (nucleotides 362-367), was considered less likely. Their prediction appears to be correct: the AUG at nucleotide 388 is followed by anopen reading frame of amino acids, whereas the AUG at nucleotide 378 is followed by an open reading frame of only 20 amino acids, ending at the UAG at nucleotide 441. Analysis of mutations thataffect the 0.3 protein suggests that
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