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Figure 4.
Fig. 4. ssSir2 mediates transcriptional silencing in vitro. (A)
Transcription assays were performed on a template containing the
T6 promoter of SSV1. Assay conditions were as described (13) (B)
Transcription reactions were assembled with recombinant,
nonacetylated (r), or acetylated (Ac) Alba. (C) Transcription
reactions were assembled containing recombinant Alba with the
indicated point mutations. (D) Transcription assays programmed
with a plasmid containing the T6 promoter, supplemented with 1
µg of ssSir2 and/or 200 µM of NAD as indicated. (E)
Upper panel: Transcription assays containing 200 µM of NAD
were assembled on the T6 promoter template and incubated with Ac
Alba in the presence of 1 µg of either ssSir2 or ssSir2
H116Y for 20 min at 65°C before the initiation of
transcription by addition of NTPs to 200 µM. Lower panel:
Transcription assays were assembled on the T6 promoter in the
presence or absence of 2.5 µg of acetylated Alba.
Reactions were supplemented with NAD to 200 µM and/or
ssSir2 [wild type (wt) or H116Y (H-Y)]. Reactions were
preincubated as above.
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