Alteration of an amino acid residue outside the active site of the ricin A chain reduces its toxicity towards yeast ribosomes.
Yeast transformants containing integrated copies of a galactose-regulated, ricin toxin A chain (RTA) expression plasmid were constructed and used in an attempt to isolate RTA-resistant yeast mutants. Analysis of RNA from mutant strains demonstrated that approximately half contained ribosomes that had been partially modified by RTA, although all the strains analysed transcribed full-length RTA RNA. The mutant strains could have mutations in yeast genes giving rise to RTA-resistant ribosomes or they could contain alterations within the RTA-encoding DNA causing production of mutant toxin. Ribosomes isolated from mutant strains were shown to be susceptible to RTA modification in vitro suggesting that the strains contain alterations in RTA. This paper describes the detailed analysis of one mutant strain which has a point mutation that changes serine 203 to asparagine in RTA protein. Although serine 203 lies outside the proposed active site of RTA its alteration leads to the production of RTA protein with a greatly reduced level of ribosome modifying activity. This decrease in activity apparently allows yeast cells to survive expression of RTA as only a proportion of the ribosomes become modified. We demonstrate that the mutant RTA preferentially modifies 26S rRNA in free 60S subunits and has lower catalytic activity compared with native RTA when produced in Escherichia coli. Such mutations provide a valuable means of identifying residues important in RTA catalysis and of further understanding the precise mechanism of action of RTA.
Gould
J H
JH
Department of Biological Sciences, University of Warwick, Coventry, West Midlands, England.
Hartley
M R
MR
Welsh
P C
PC
Hoshizaki
D K
DK
Frankel
A
A
Roberts
L M
LM
Lord
J M
JM
D000596:Amino Acids / Q000235:genetics*; D001665:Binding Sites; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D005804:Genes, Lethal; D010957:Plasmids; D011687:Purines / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism*; D012276:Ricin / Q000096:biosynthesis / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
D000596:Amino Acids; D004271:DNA, Fungal; D011687:Purines; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D012276:Ricin; C030985:purine