FIGURE 6.
Competition of
,
tetracycline, and hygromycin B binding to the mRNA-70 S ribosome complex with
YoeB, and release of the 3′-end portion of the ompA mRNA
cleaved by the addition of YoeB from ribosomes. A, effect of YoeB
on the formation of the ompA mRNA-70 S
complex. Lane 1, without YoeB(His)6; lane 2, 20.7
nm YoeB(His)6; lane 3, 1 μm
and 0.05
μm 70 S ribosomes; lanes 4–10, 1 μm
and 0.05
μm 70 S ribosomes and 20.7, 10.4, 2.58, 0.64, 0.16, 0.04, and
0.01 nm YoeB(His)6, respectively. B, effect of
on the
activity of YoeB. Lane 1, without YoeB(His)6; lane
2, 20.7 nm YoeB(His)6; lane 3, 1
μm
and 0.05
μm 70 S ribosomes; lane 4, 0.05 μm 70 S
ribosomes and 20.7 nm YoeB(His)6; lanes
5–10, 0.05 μm 70 S ribosomes and 20.7 nm
YoeB with 1, 4, 8, 16, 32, and 64 μm
,
respectively. C, binding of
to 70 S ribosomes at 37 °C in the absence and presence of different
amounts of YoeB(His)6. 70 S ribosomes (0.05 μm) were
first incubated with
(1 μm) and the ompA mRNA (0.035 μm) for
10 min at 37 °C, then 0, 0.04, 0.64, 2.58, and 10.4 nm YoeB was
added. The reaction mixtures were incubated for an additional 10 min at 37
°C and then applied to nitrocellulose filters (Millpore 0.45
μm HA), which were washed twice with 2 ml of buffer A before
measuring the radioactivity. D, toeprinting experiment with the
ompA mRNA and
.
Lane 1, without YoeB(His)6; lane 2, 20.7
nm YoeB(His)6; lane 3,
;
lane 4, 1 μm
and 0.05 μm 70 S ribosomes; lane 5, 1 μm
,
0.05 μm 70 S ribosomes, and 20.7 nm
YoeB(His)6; lane 6, 1 μm
and 0.05
μm 70 S ribosomes; lane 7, 1 μm
,
0.05μm 70 S ribosomes, and 20.7 nm
YoeB(His)6. E, release of the 3′-end portion of the
ompA mRNA cleaved by the addition of YoeB from ribosomes. Lane
1, without YoeB(His)6; lane 2, 20.7 nm
YoeB(His)6; lanes 3 and 4, with 1
μm
and 0.05
μm 70 S ribosomes; lanes 5 and 6, 1
μm
and 0.05
μm 70 S ribosomes and 20.7 nm YoeB(His)6.
Lanes 3 and 5, the samples were centrifuged at 90,000
× g for 1 h at 4 °C, and the supernatant was taken for the
primer extension. F, effect of YoeB on the 30-base RNA binding to the
S
ribosome complex. 70 S ribosomes (0.05 μm) were first incubated
with 0, 42, 168, 336, and 672 nm YoeB(His)6 and
(1
μm) for 10 min at 37 °C. The 5′-end labeled 30-base
RNA (35 nm) was then added. The reaction mixture was incubated for
an additional 10 min at 37 °C, and then applied to nitrocellulose filters
(Millpore 0.45 μm HA), which were washed twice with 2 ml of
buffer A before measuring the radioactivity. G, effect of
tetracycline on the activity of YoeB-mediated mRNA cleavage. Lane 1,
1 μm
and 0.05
μm 70 S ribosomes; lane 2, 1 μm
, 0.05
μm 70 S ribosomes, and 160 μm tetracycline;
lane 3, 1 μm
, 0.05
μm 70 S ribosomes, and 10.4 nm YoeB(His)6;
lane 4, 1 μm
, 0.05
μm 70 S ribosomes, 10.4 nm YoeB(His)6, and
160 μm tetracycline. H, effect of hygromycin B on the
activity of YoeB-mediated mRNA cleavage. Lane 1, 1 μm
and 0.05
μm 70 S ribosomes; lanes 2 and 3, 1
μm
, 0.05
μm 70 S ribosomes, and 0.64 and 1.6 mm hygromycin B,
respectively; lane 4, 1 μm
, 0.05
μm 70 S ribosomes, and 20.7 nm YoeB(His)6;
lanes 5 and 6, 1 μm
, 0.05
μm 70 S ribosomes, 20.7 nm YoeB(His)6, and
0.64 and 1.6 mm hygromycin B, respectively. SD,
Shine-Dalgarno; FL, full-length.