Protein splicing involving the Saccharomyces cerevisiae VMA intein. The steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing system.
Protein splicing involves the excision of an internal protein segment, the intein, from a precursor protein and the concomitant ligation of the flanking N- and C-terminal regions. It occurs in mesophilic bacteria, yeast, and thermophilic archaea. The ability to control protein splicing of a thermophilic intein by temperature and pH in a foreign protein context facilitated the study of the mechanism of protein splicing in thermophiles. On the other hand, no direct studies have been done on the mechanism of protein splicing in mesophiles. We examined the splicing of a chimeric protein containing the intein of the vacuolar ATPase subunit (VMA) of Saccharomyces cerevisiae that involves cysteines rather than serines at the reaction center. The steps in the splicing process were deduced by analyzing intermediates and side products that accumulated as a result of amino acid substitutions and were found to be analogous to those occurring in thermophiles. Moreover, appropriate amino acid replacements allowed us to develop the first mesophilic in vitro protein splicing system as well as strategies for modulating the rate of protein splicing and for converting the splicing reaction to an efficient protein cleavage reaction at either splice junction.
Chong
S
S
New England Biolabs, Beverly, Massachusetts 01915, USA.
Shao
Y
Y
Paulus
H
H
Benner
J
J
Perler
F B
FB
Xu
M Q
MQ
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D017124:Conserved Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases