![abi time clock abi time clock](https://i.pinimg.com/originals/fb/c7/3f/fbc73fb11a163bafb22be44282c0b277.jpg)
) prompted us to examine nondenatured extracts to determine whether evidence of a clock protein complex involving one or both of the FRQ forms could be found. The estimated sizes of both IVTFRQ and both forms of endogenous FRQ after λPPase treatment are 135 and 125 kDa ( Figure 3C), still appreciably larger than the predicted sizes of 108 and 97 kDa, suggesting that aspects of the FRQ primary ( This indicates that the upper FRQ band represents the full-length protein with a mobility indistinguishable from full-length in vitro-translated FRQ (IVTFRQ Figure 3B), in addition to showing that the alternative initiation described above does not require Neurospora-specific factors. Treatment of in vitro-translated FRQ with phosphatase also generates two bands with mobilities indistinguishable from the two bands observed for endogenous phosphatase-treated FRQ. The increased mobility of FRQ is fully inhibited by sodium vanadate, indicating that the change in mobility is not due to the activity of contaminating proteases ( Figure 3B). Upon incubation with λPPase, FRQ is converted to two distinct forms at all times of the day, with the lower mobility band likely corresponding to FRQ 1–989 and the higher mobility band to FRQ 100–989.
#Abi time clock series#
In samples not subjected to immunoprecipitation and incubation with λPPase ( Figure 3A, lanes 1–6), at least five bands were resolved for the upper FRQ series and three bands for the lower FRQ series. λPPase removes phosphate residues on serine, threonine, tyrosine, and histidine residues and is fully inhibited by vanadate. To determine if the size discrepancy is due to phosphorylation, FRQ from circadianly timed extracts was subjected to immunoprecipitation followed by treatment with lambda protein phosphatase (λPPase New England Biolabs) and Western blot analysis ( Figure 3A). Furthermore, the estimated sizes of the two series of bands range from 140–160 kDa for FRQ 1–989 and 125–140 kDa for FRQ 100–989, and deviate greatly from the predicted sizes of 108 and 97 kDa. The mobilities of both FRQ 1–989 and FRQ 100–989 show striking changes over the circadian day, with the minimum mobility immediately preceding the disappearance of FRQ ( Figure 3).
![abi time clock abi time clock](https://i.pinimg.com/originals/ad/93/eb/ad93ebf6121e138a0a8249ffea12fdf7.jpg)
Also, the AUGs at codons #1 and #100 have good consensus sequences for translational initiation (while the AUG at codon #11 does not) ( The predicted difference in size for the proteins initiated at AUG#1 or AUG#2 and AUG#3 is ∼10 kDa, consistent with the differences in sizes observed by Western blot. These results clearly demonstrate that translational initiation in the frq ORF can occur at more than one position. In FRQ ΔAUG#1and#2 (lacking the first two AUGs in the FRQ ORF), only the lower molecular weight series of bands are produced in FRQ AUG#3→GAU (lacking the third AUG), only the higher molecular weight series of bands are produced and in FRQ ΔAUG#1,#2,and#3→GAU, both series of bands are eliminated. In the wild-type strain, both series of bands are observed. The constructs were introduced into the frq 10 (null) strain and assayed for expression of FRQ by Western blot ( Figure 2B). To determine if the clusters of FRQ bands are due to choice of translational initiation sites, we selectively eliminated some or all of the potential initiation sites ( Figure 2A).