Mutation and modeling analysis of the Saccharomyces cerevisiae Swi6 ankyrin repeats.
The Swi4-Swi6 family of transcription factors confers G1/S specific transcription in budding and fission yeast. These proteins contain four ankyrin repeats, which are present in a large number of functionally diverse proteins and have been shown to be important for protein-protein interaction. However, no specific sequence has been identified that is diagnostic of an ankyrin repeat-interacting protein. To determine the function of the ankyrin repeats of Swi6, we generated both random and site-directed mutations within the ankyrin repeat domain of Swi6 and assayed the transcriptional function of these mutant swi6 alleles. We found six single mutations, scattered within the first and the fourth repeats, that generate a temperature-sensitive Swi6 protein. In addition, we found that alanine substitutions for the most conserved residues in each repeat were highly deleterious and also confer temperature sensitivity. Most of these swi6 alleles are able to form ternary complexes with Swi4 and DNA, but these complexes display reduced mobility in band-shift gels, suggesting a dramatic conformational change. We have modeled the ankyrin repeats of Swi6 using the coordinates derived for 53BP2 and find that, despite its low level of sequence conservation, these modeling studies and our mutation data are consistent with Swi6 having a structure very similar to that of 53BP2. Moreover, all but one of our single mutants and all of the site-directed mutants disrupt critical structural features of the predicted folding pattern of these repeats. We conclude that the ankyrin repeats play a major structural role in Swi6. Ankyrin repeats are unlikely to have inherent protein or DNA binding properties. However, they form a characteristic and stable structure with surfaces that may be tailored for many different macromolecular interactions.
Ewaskow
S P
SP
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, and Department of Biology, Lewis and Clark College, Portland, Oregon 97219, USA.
Sidorova
J M
JM
Hendle
J
J
Emery
J C
JC
Lycan
D E
DE
Zhang
K Y
KY
Breeden
L L
LL
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017089:Ankyrin Repeat / Q000235:genetics*; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins / Q000737:chemistry; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D015151:Immunoblotting; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics*; D014158:Transcription, Genetic
D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C083209:MBP1 protein, S cerevisiae; C062214:SWI4 protein, S cerevisiae; C075111:SWI6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C090468:TP53BP2 protein, human; D014157:Transcription Factors; D000409:Alanine