jbc
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2 m M Mg 2+ and 400 μM Zn 2+ 417 - 311 - 249 - 200 - 151 - 118 - 100 - 82 - 66 - 48 - 40 - 726 - tim e M -E 2 m M Mg 2+ 400 μM Zn 2+ tim e tim e Prim er 2 m M Mg 2+ and 400 μM Zn 2+ 417 - 311 - 249 - 200 - 151 - 118 - 100 - 82 - 66 - 48 - 40 - 726 - tim e M -E 2 m M Mg 2+ 400 μM Zn 2+ tim e tim e Prim er Supplemental Figure. Extension by HIV-RT with different divalent cations on an unstructured template. 5‘- 32 P-radiolabeled oligo-dT 20 primer was hybridized 1:10 (w:w) to poly-rA template and added at a final concentration of 0.002 μg/μl in rA to a reaction mixture containing 100 μM dTTP, 50 mM Tris-HCl (pH=8), 80 mM KCl, 1 mM DTT, and either 2 mM MgCl 2 , 400 μM ZnCl 2 , or both. To initiate reactions, RT was added at a final concentration of 25 nM. Aliquots were removed and extension stopped with 2X formamide-dye at 5,15, 30 seconds, 1, and 2 minutes for MgCl 2 -only reactions or 5, 15, 30 seconds, 1, 2, 4, 8, and 16 minutes for reactions containing ZnCl 2 . The maximum extension rate was ~25 times faster in the presence of MgCl 2 than ZnCl 2 (~25 nt/sec and ~1 nt/sec, respectively). Extension in the presence of both cations is indistinguishable from ZnCl 2 -
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2 mM Mg2+ and 400 μM Zn2+
417 -311 -249 -200 -
151 -
118 -
100 -
82 -
66 -
48 -
40 -
726 -
timeM -E
2 mM Mg2+ 400 μM Zn2+
time time
Primer
2 mM Mg2+ and 400 μM Zn2+
417 -311 -249 -200 -
151 -
118 -
100 -
82 -
66 -
48 -
40 -
726 -
timeM -E
2 mM Mg2+ 400 μM Zn2+
time time
Primer
Supplemental Figure. Extension by HIV-RT with different divalent cations on an unstructured template. 5‘-32P-radiolabeled oligo-dT20 primer was hybridized 1:10 (w:w) to poly-rA template and added at a final concentration of 0.002 μg/μl in rA to a reaction mixture containing 100 μM dTTP, 50 mM Tris-HCl (pH=8), 80 mM KCl, 1 mM DTT, and either 2 mM MgCl2, 400 μM ZnCl2, or both. To initiate reactions, RT was added at a final concentration of 25 nM. Aliquots were removed and extension stopped with 2X formamide-dye at 5,15, 30 seconds, 1, and 2 minutes for MgCl2-only reactions or 5, 15, 30 seconds, 1, 2, 4, 8, and 16 minutes for reactions containing ZnCl2. The maximum extension rate was ~25 times faster in the presence of MgCl2 than ZnCl2 (~25 nt/sec and ~1 nt/sec, respectively). Extension in the presence of both cations is indistinguishable from ZnCl2-only reactions. Lane M, size marker in nucleotides; land –E, no enzyme added.
