SRP RNA distal end triggers GTP hydrolysis **
5.5.6 GTPase assay
GTPase rate constants were determined using a GTPase assay. In general, reactions contained 100 nM Ffh, 200 nM SRP RNA (wild-type or mutants), and varying concentrations of FtsY were incubated with 100 µM GTP (doped with γ-32P-GTP).
Reactions were quenched by 0.75 M KH2PO4 (pH 3.3) at different time points, separated by thin layer chromatography (TLC), and quantified by autoradiography.
5.6 Acknowledgements
We thank members of the Shan group for helpful comments on the manuscript.
This work is supported by NIH grant GM078024 to S.-o.S., an NIH instrument
supplement to grant GM45162 to D.C. Rees, and Caltech matching fund 350270 for the single-molecule instruments. S.-o.S. was supported by the David and Lucile Packard Fellowship in science and engineering, and the Henry Dreyfus teacher-scholar award.
5.7 Figures and figure legends
Figure 5.1 General scheme of the function of the SRP RNA during co-translational protein targeting. (A) Working model of co-translational protein targeting by SRP. See main text for introduction. (B) The secondary structure of the E. Coli SRP RNA. Four different domains are noted with different colors.
SRP
FtsY
SecYEG RNC
Signal
Sequence SRP RNA
Ffh-NG Ffh-M Ribosome mRNA
T
T
T T T TT
D
D
Cytosol Periplasmic space
'''''#5#5'55''55
###!#####'''!#''5#'!
# 5'5
5###'
!'''#
#!
#'5'
!#'#
5'#' 5!#
#!'5 5#5'555
!'!#''!
!##!
! ''5
##'
#!''
!#'!
5##
!'' ''
!!
A E D C B
I IIIII.III. . .. I I IIIII IIII IIII..I II. III
wildtype E. Coli SRP RNA
A
B
1. 11. 21. 31. 41. 51
.
61. 71.
81. 91. 101. 111
.
1
2
3 4
!Linker
Tetraloop Ffh-M binding site Distal-end
docking site
Catalytic site
Figure 5.2 An intact SRP RNA distal end is required for efficient GTP hydrolysis.
(A) GTPase assay showing the function of the SRP RNA. The kcat/Km and kcat values are 164 µM-1•min-1 and 90 min-1 for +SRP RNA curve, 0.69 µM-1•min-1 and 8.5 min-1 for – SRP RNA curve. (B) Single-nucleotide truncation mutants of 92mer and their GTPase activity relative to wild-type SRP RNA and 82mer.
' 5 5 ' ' 5
# ' ' 5 # '• • • • 92mer
10• 15
•
100•
' 5 5 ' ' 5 ' ' 5 # '• • • • 91mer ' 5 5 ' ' 5 ' 5 # '• • • 90mer ' 5 5 ' ' 5 5 # '• • 89mer ' 5 5 ' ' 5 # '• 88mer ' 5 5 ' ' 5 '• 87mer 5 '• 82mer 0
0.2 0.4 0.6 0.8 1
wt
92mer91mer90mer89mer88mer87mer82merNo RNA Relative kcat
SRP RNA mutant Stimulated GTPase activity
A
0 20 40 60 80 100
0 2 4 6 8 10 25 50
FtsY (MM) kobsd (min-1)
Stimulated GTPase activity
TSRP + FtsYT ! TSRP•FtsYT " DSRP•FtsYD
+SRP RNA
–SRP RNA slope = kcat/Km plateau = kcat
B
Figure 5.3 Base-specificity of the RNA stimulatory effect. (A-D) Point-mutagenesis of specific SRP RNA distal-end bases and their GTPase activity. Four nucleotides with most defective mutants are shown: G14 (A), U15 (B), G96 (C), and U98 (D). (E)
wt defective active
E
# 5 ' 5 5 ' ' 5 5 ' ! # ' ' 5 # ' !
#
5
• • • •
C(0.91) G(2.3) C(2.5) A(0.83)
8 9 10 11 12 13 14 15 16
95 96 97 98 99 100 101 102 103
U(0.97) C(0.23) A(0.74)
U(0.12) C(0.11) A(0.13)
G(0.26) C(0.25) A(0.23)
A(0.15) C(0.15) U(0.18) A(0.15)
G(1.6) U(2.3) A(0.26) C(0.38) G(0.51) A(3.0) C(0.76) U(1.7) A(0.96) U(0.87)
G(0.56) 0
20 40 60 80 100
0 5 10 15 20
FtsY (MM) kobsd (min-1)
GTPase activity of G14 mutants
wt G14A G14C G14U
••
•• k
obsd (min-1)
wt U15A U15C U15G
••
•• GTPase activity of U15 mutants
A B
0 20 40 60 80 100
0 5 10 15 20
FtsY (MM)
0 20 40 60 80 100
0 5 10 15 20
FtsY (MM)
GTPase activity of G96 mutants
wt G96A G96C G96U
••
••
C D
0 20 40 60 80 100
0 5 10 15 20
FtsY (MM)
GTPase activity of U98 mutants U98C
•U98G
•
Activity scale Summary of GTPase activity of SRP RNA mutants
kobsd (min-1) kobsd (min-1)
wt
•U98A
•
F
0 20 40 60 80 100
0 5 10 15 20
FtsY (MM) kobsd (min-1)
GTPase activity of double mutants
wt G13U&U98G U15G&G96U G14U&C97A
••
••
0.25!
0.35!
0.31!
Summary of the GTPase activity of the point-mutants at the distal-end docking site. (F) GTPase activity of base-pair mutants.
Figure 5.4 Loop E controls the stimulatory activity of the SRP RNA. (A) Design of loop E mutants. E-1, E+1, and E+2 create shrinking and expansion of loop E. Ecg tightens the loop E by replacing the UA pairs with CG pairs. ΔE, ΔE+1, and cE eliminate
A
wildtype 5 5
! !
# 5'5
E 5 5
! !
#
55 E-1
5 5
! !
#
#5'5 E+1
# # ' '
#
5'5 Ecg
Relative kcat
Loop E mutant wt E-1 E+1 Ecg
No RNA 5 5
! !
#
##5'5 E+2
$E 5 5
! !
# '
cE 5 5
! !
!#! 5'5
$E+1 5 5
! !
# '5
B
E-1 E+1
$E+1E+2
••
••
Wildtype
0 20 40 60 80 100
0 1 2 3 4 5 20
FtsY (MM) kobsd (min-1)
GTPase activity of loop E mutants (1)
0 20 40 60 80 100
0 5 10 15 20
FtsY (MM)
GTPase activity of loop E mutants (2)
kobsd (min-1) Wildtype
Ecg$E cE
••
•
0 0.2 0.4 0.6 0.8 1
E+2 $E
$E+1 cE
C Summary of GTPase activity of loop E mutants
loop E. (B) GTPase activity of the loop E mutants in (A). (C) Summary of the relative GTPase rate of the loop E mutants.
Figure 5.5 Loop E mutants cause defect in the docking of the GTPase complex at the distal end of the SRP RNA. (A) Single-molecule setup for determining the migration of the Ffh-NG/FtsY-NG complex along the SRP RNA scaffold. Ffh C153 is labeled with Cy3. The 3’-end of the SRP RNA is labeled with Quasar670. (B) Fluorescent signals (upper panel) and FRET trajectory (lower panel) of the SRP-FtsY complex in GppNHp.
Hidden Markov Modeling (HMM) of the FRET trajectory is shown in navy. (C) Sample FRET trajectories (cyan) and HMM simulation (navy) of E-1, E+1, and Ecg SRP RNA mutants (left panel). The histograms for the FRET traces are shown in the right panel.
(D) Correlation between the percentage of high FRET state and the GTPase activity.
Standard curve (solid line) is the linear fit of three data points, wild-type SRP RNA, 82mer, and 99A (black crosses). E-1, E+1, and Ecg data points are shown in red cycle.
C
0 0.05 0.1
0 0.5 1
0 0.5 1
0 0.5 1
0 5 10 15 20 25 30
FRET
Histogram
Time (s) Frequency
E-1
E+1
Ecg
0 10 20 30 40 50
0 1 2 3
% High FRET
Relative GTPase activity Wildtype
99A
82mer E-1 Ecg
E+1
D
r = 0.985 PEG
Neutravidin DNA splint
SRP RNA
Biotin Quasar670
Coverslip Ffh-M
FtsY-NG Ffh-NG
Cy3 Proximal state
Low FRET
Distal state High FRET
TIRF excitation
Tetraloop end
Distal end
A B
0 0.5 1 0 500 1000
0 5 10 15 20 25 30
Fluorescence intensity (A.U.)FRET
Time (s) – Donor (Cy3)
– Acceptor (Quasar670) – FRET (exp) – FRET (HMM)
Figure 5.6 Catalytic bases do not contribute to the docking. (A) Crystal structure of the SRP-FtsY complex at the distal state. Shown in yellow is the protruding base that inserts into the Ffh-NG/FtsY-NG interface. (B) Secondary structure of the SRP RNA loop D. (C) Single-molecule traces (left panel) and histograms (right panel) of G83A and C86G mutants.
0 0.5
1 G83A
0 0.05 0.1
0 5 10 15 20 25 30
Time (s) Frequency
0 0.5
1 C86G
FRET
C Histogram
A
# ' ! # ' # 5 '
#!
#'5' D
21.
81. 83. 86.
FtsY-NG Ffh-NG
Ffh-M SRP RNA
Tetraloop Distal-end
docking site
Catalytic nucleotide
B
Figure 5.7 Auxiliary docking interaction mediated by C87. (A) GTPase activity of C87 mutants. (B) Single-molecule trace (left panel) and histogram (right panel) of C87A mutant. (C) Correlation between the percentage of high FRET state and the GTPase activity. Standard curve (dashed line) is the linear fit of the six data points in Figure 6D (black crosses). G83A, C86G, and C87A data points are shown in colored cycle.
0 50 100 150 200
0 5 10 15 20
FtsY (MM)
wt C87A C87G$C87
••
•• kobsd (min-1)
GTPase activity of C87 mutants
A
% High FRET
Relative GTPase activity
C
0 10 20 30 40 50
0 1 2 3
C87A G83A
C86G wildtype
B
FRET
0 0.5
1 C87A
0 0.05 0.1
0 5 10 15 20 25 30
Time (s) Frequency
Histogram
Figure 5.8 C87 acts independently of the distal-end docking site. GTPase activity of the SRP RNA mutants with combined active mutation. C97U and G99A are active mutations at the distal-end docking site. C87A is the active mutation at the auxiliary docking site.
0 100 200 300 400
0 5 10 15 20
FtsY (MM) Wildtype C87A&G99A
C87A&C97U C97U&G99A
kobsd (min-1) 5.5!4.6!
3.0!
GTPase activity of double mutants