Abstract
ANDREWS, ANDREW JOSEPH. Characterization of the Methanococcus jannaschii
RNase P holoenzyme. (Under the direction of J.W. Brown)
The RNase P RNAs from Methanococcus and Archaeoglobus lack secondary structural
features essential for the recognition of pre-tRNA. In the absence of protein, these RNase
P RNAs lack catalytic activity. How the Methanococcus and Archaeoglobus RNase P
holoenzymes compensate for the absent RNA structural elements is not known, but we
hypothesize that it is via additional proteins. The Methanococcus jannaschii RNase P
holoenzyme has been purified for structural and functional characterization. This enzyme
has a buoyant density in Cs2SO4 of 1.39 g/ml and an apparent molecular weight of greater
than 400kDa. The holoenzyme has a Km of 68 nM, a kcat of 37nM min-1 (similar to that
of other RNase P enzymes) and tolerates a wide range of ionic conditions. Six potential
protein subunits have been identified on the basis of copurification with enzymatic
activity. The molecular weight of three of these bands is consistent with apparent
holomologs of RNase P proteins from Methanobacterium thermoautotrophicum and
Saccharomyces cerevisiae.
Characterization of the Methanococcus jannaschii RNase P holoenzyme.
by
Andrew Joseph Andrews
A thesis submitted to the Graduate Faculty of
North Carolina State University
in partial fulfillment of the
requirements for the Degree of
Masters of Science
Microbiology
Raleigh
2000
APPROVED BY:
Chair of Advisory Committee
Biography
1.
1992 Graduated from Broughton High School, Raleigh, N.C.
2.
1994 Received The North Carolina Emergency Medical Technician
Paramedic
3.
1995
4.
1998 Graduated North Carolina State University, Raleigh N.C.
Degree: B.S. Microbiology
Employed as a half-time teacher for Wake County School System
ii
Table of Contents
List of Tables
iv
List of Figures
v
Introduction
Bacterial RNase P
Eukaryotic Nuclear RNase P
Archaeal RNase P
Statement of Purpose
1
1
2
5
7
Materials and Methods
RNase P Activity Assays
Purification
Cultivation of M. jannaschii
Preparation of Cleared Lysate
DEAE Sepharose Chromatography
Cs2SO4 Buoyant Density Gradient Centrifugation
Q-Sepharose Chromatography
Glycerol Gradient Centrifugation
Affinity Purification
Protein Gels and Quantification
Northern Blots
Concentration of RNase P Samples
Size Exclusion
8
8
9
9
9
11
11
11
12
12
13
15
15
16
Results
16
16
22
26
26
Purification
The Effects of Monovalent and Divalent Salts on RNase P Activty
The Effects of Temperature on RNase P Activity
Enzyme Kinetics
Discussion
Purification of the Methanococcus jannaschii RNase P holoenzyme
Affinity Purification of RNase P
Fold Purification
Evaluating the Size of Methanococcus jannaschii RNase P
Enzymatic Differences between the Methanogenic Archaeal RNase P
Holoenzymes
Proteins Purifying with Methanococcus jannaschii RNase P Holoenzyme
Future Research Areas
26
26
29
30
30
References
41
Appendices
Appendix 1. TP/PT Construct
44
32
33
39
iii
List of Tables
1.
Fold Purification
2.
Molecular Weight of Bands Purifying with RNase P
Page
31
38
iv
List of Figures
Page
1.
Color Coded Secondary Structure of E.coli
3
2.
Secondary Structures
4
3.
Flow Chart of Purification
10
4.
Affinity Column Results
14
5.
DEAE-Sepharose Chromatrography
18
6.
Cs2SO4 Gradient
19
7.
Q-Sepharose Chromatrography
20
8.
Glycerol Gradient
21
9.
MgCl2 Titration
23
10.
Divalent Ion Exchange
24
11.
NH4OAc Titration
25
12.
Effects of Temperature on RNase P Activity
27
13.
Lineweaver-Burk Plot
28
14.
Alignment of archaeal homologs of the Yeast RNase P
subunit Pop5p
34
Alignment of archaeal homologs of the Yeast RNase P
subunit Rpr2p
35
Alignment of archaeal homologs of the Yeast RNase P
subunit Rpp1p
36
Alignment of archaeal homologs of the Yeast RNase P
subunit Pop4p
37
18.
Secondary Structures for TP/PT Constructs
45
19.
Results from TP assays
46
15.
16.
17.
v
Introduction
RNase P is the enzyme responsible for removing the 5′ end of precursor-tRNA and other
small RNAs (1). It is an ubiquitous enzyme found in all three branches of life as well as
intracellular organelles that synthesize tRNA (2). RNase P is a ribonucleoprotein in all
characterized instances, and it is the RNA, not the protein, that is the catalytic subunit of
the enzyme (2). The catalytic reaction carried out by the RNase P RNA leaves a 3′
hydroxyl on the leader fragment and a 5′ phosphate on the now mature length tRNA (2).
The fact that bacterial and some archaeal RNase P RNAs are by themselves capable of
binding, cleaving and then releasing substrate multiple times makes this a true RNA
enzyme (3).
Bacterial RNase P
Bacterial RNase P consists of an RNA (ca. 140kDa) and a single small protein (14kDa
(4). The RNA and the protein have a one to one stoichiometry in the holoenzyme and
each component is encoded by single copy genes that are required for cell viability (5,6).
These components can be recombined in vitro to reconstitute the active holoenzyme (7).
In B. subtilis the protein subunit does not change the MgCl2 concentration required for
the cleavage reaction but it does change the interaction of the enzyme with pre-tRNA(8).
The 5′ end of the pre-tRNA has been shown to bind a cleft in the B. subtilis RNase P
protein (9,10). Removing the entire region of RNase P RNA distal to the start of P7 will,
in the right ionic conditions, result in a non-specific ribonuclease activity (11). This same
RNA, in the presence of the protein component, will correctly cleave substrate with a
1
100-fold increase in Km, but it retains a similar kcat. The protein also broadens the
enzyme’s specificity and reduces the effect of product inhibition (12).
The RNA from all known RNase Ps share a common secondary structural core (4,13,14).
It is well established that the RNA component of RNase P in Bacteria is catalytically
active in vitro (3). RNase P RNA helix P4 coordinates the Mg++, that carries out a
hydroxyl attack on the scissile phosphate of the pre-tRNA cleavage site (13,15-18).
There are two additional regions of the RNA that are implicated in the recognition of the
pre-tRNA substrate (Figure 1). The CCA on the 3′ end of the pre-tRNA binds the L15
loop in bacterial RNase P RNA (19). Mutation of the CCA binding region in L15
disrupts the substrate affinity of the RNA-alone reaction in both E. coli and B. subtillis
unless the compensatory changes are made in the pre-tRNA (19). The P8 has been
identified in cross-linking studies as interacting with the T-stem loop of tRNA (20).
Outside of the catalytic region, P8 and L15 of RNase P RNA are regions identified in
chemical modification and cross-linking studies that interact with the pre-tRNA
(1,19,20).
Eukaryotic Nuclear RNase P
The eukaryotic nuclear RNase P holoenzyme differs greatly from that of Bacteria (Figure
2). The RNase P holoenzyme in Saccharomyces cerevisiae consists of an RNA and nine
protein subunits (21). Eight of these nine proteins are also found in RNase
2
160
CA
A
G
C G
G C
C G
C G
C GA
G U
G C
AU A
GC G
C G
G UA
C GA
140
180
GGU
AC
G
A
A
A
A
GU A
C
A
A
G
CG
G
G
U
A
G
GG
C A
G
AC G G
A
CC
G
G 200
A
CG
CG
C
G C
UGG
GU
A
GC
G
U
A
C G
AC A
C
C
220 U
A
A
G
C
U
C A G A A A U GG
CA
C
A C C C AC G U
240
A
C
A AUA
120 C A
A GGG
260
A
CC
G
G
G
GCC
C
G
G
A
G
C
A
A
G
G
G
AUA
G GG
UUC
G C U GG G
A
C
CCUC
U CG G
U
CCC
G
A
G 300 A
A
G
A
C A
A
G
G
G
C
UGG
C
U
U
C G
U 80 G
G
U
C
G
A
G
G A
G
A
CG
U
U
G
C
U
C
G
100
A A
G CG
A
280
C
CC
U
G C C A G U G A GC
G
A
C GGU C GU U AG
A
C
320
U
A
A
G
G
60 G
A
A
U
G
G G G G G A GA C G G C G G A G G G G
G
C
A
U C U C C U CU GC U GC U UC C C
A
U
40
G
U
G
G
C G
20
U A
G C
A U
1
C G 340
A U
GA A GC UGA C C A G C C A C G A C A
G
C U U U GA C U GG U
A
C A
A
U U C G G C C CA
C
C
360
U
5' binding region
P12
Escherichia coli
RNase P RNA
P13
P14
P9
P7 P5
L15
P16
P17
P18
P8
P6
P4
P3
P2
P1
T stem-loop binding region
3' CCA binding region
Figure 1. Secondary structure of E.coli RNase P RNA with substrate binding regions
color-coded. Helices are labeled P1-P18 more information can be found on the RNase P
database: www.mbio.ncsu.edu/RNaseP/home/html (4).
3
A.
B.
C.
D.
Figure 2. RNase P RNA Secondary Structures for A. Methanococcus jannaschii (M-type
archaea), B. Methanobacterium thermoautotrophicum (A-type archaea), C.
Saccharomyces cerevisiae D. Micro P- minimum RNase P RNA that can carry out the
RNA alone reaction. Additional secondary structures are available on the RNase P
database at www.mbio.ncsu.edu/RNaseP/home.html (4).
4
MRP, an enzyme of the rRNA processing pathway (21). RNase MRP and RNase P in the
eukaryotes also share a similar RNA structure in helices P1, P2, P3, P4, and P19 (22).
Reconstitution of the Saccharomyces cerevisiae RNase P from its separate subunits has
not been accomplished. The human RNase P has been shown to contain homologs of at
least some of the proteins found in yeast (23) (21).
Eukaryotic RNase P RNAs have no demonstrable activity in the absence of protein and
limited structural and sequence similarites to their bacterial counterparts (4,22). As more
sequences have become available (now a total of 23 nuclear sequences), of the eukaryotic
RNase P RNA, the resolution of the secondary structure has increased. Homologs of P1,
P2, P3, P4, and P10/11, the catalytic core of RNase P have been identified (24). It has
been suggested that the functional role of the lost L15 in substrate CCA recognition has
been taken over by an internal loop in P3(13). In the absence of characterized substrate
recognition and structural stabilizing elements, our understanding of the RNA eukaryotic
RNase P RNA secondary structure and function have lagged behind that of Bacteria.
Adding to the difficulty of defining the eukaryotic RNase P RNA secondary structure is
the limited amount of sequence data and the extreme (by comparison to Bacteria)
variability of the sequences.
Archaeal RNase P
The RNase P RNAs from some groups of the Archaea were recently shown to be
catalytically active (25). However, the active archaeal RNase P RNAs are very
inefficient compared to those of Bacteria. They require extremely high monovalent and
5
divalent salt (3M ammonium acetate and 300mM MgCl2) concentrations in conjunction
with prolonged incubation times at higher temperatures(25). Prior to this discovery, it
was thought that the RNA moities of these enzymes were inactive like those of the
eukaryotes. The early work on the holoenzyme of archaeal RNase P was focused on
determining if there was an RNA present in the enzyme. There are only two reports of the
characterization of RNase P holoenzymes from the Archaea: those of Haloferax volcanii
and Sulfolobus acidocaldarius (26-28). In these studies the authors showed: there is an
RNA, that the holoenzyme is active and that the density of Sulfolobus acidocalderius is
1.27g/ml in Cs2SO4 (close to that of protein alone), while the density of Haloferax
volcanii is 1.61g/ml (close to that of RNA alone) (26-28).
There are two general types of archaeal RNase P RNA structures: the M-type found in
Methanococcus and Archaeoglobus, and the A-type (or ancestral type) found in other
Archaea (personal communication K. Harris) (Figure 2). The archaeal A-type is
remarkably similar to that of A- type bacterial form. In general, the archaeal A-type
RNase P RNAs are missing the variably-present stabilizing structures of the bacterial
RNA (14,29-31). However, M-type RNase P RNAs lack P8, L15, P16, P17, and P6 and
have a unique rearrangement of the cruciform region (personal communication K.
Harris)(25). The M-type RNase P RNAs also have a single uninterrupted helix composed
of both the P10 and P11 similar to that of eukaryotic RNase P RNAs (personal
communication K. Harris). It is not surprising that M-type RNase P RNAs missing the
essential elements of substrate recognition would be catalytically inactive. Although the
6
RNase P RNA alone from Methanococcus species are inactive, RNase P activity can be
readily detected in the cell extracts.
The RNase Ps from Methanobacterium species are active in the absence of protein and
have been shown to reconstitute functional holoenzymes the with B. subtillis protein (25).
However, no protein resembling that of Bacteria has been found in any of the archaeal
genomes. On the other hand four proteins genes found in Archaea (including the genome
of Methanococcus jannaschii) have been found to have weak but significant similarity to
proteins of the yeast holoenzyme (personal communication T. Hall). The proteins from
the yeast holoenzyme are Pop5p, Pop4p, Rpr2p, and Rpp1p (21). Western blots using
antibodies against the Methanobacterium thermoautotrophicum proteins MTH687p,
MTH688p, MTH1618p, and MTH11p show them copurifying with RNase P activity
from the same organism. These antibodies can also imunoprecipitate RNase P activity
from partially-purified enzyme preparations. This suggests the archaeal RNase P
holoenzyme is a chimera of a bacterial-like RNA structure with eukaryotic-like proteins.
Statement of Purpose
The RNA world hypothesis requires that functions originally carried out by RNA have
been taken over by protein. Straightforward “proof of principle” of this process would be
examples of structural and functional roles carried out by RNA in some organisms and
protein in others. No such example is known, but the P8 and L15 of archaeal RNase P, if
replaced by protein in M-type RNase P RNA such as Methanococcus jannaschii may
7
provide such an example. The role of substrate binding once held by P8 and L15 in the
RNA subunit is hypothesized to have been replaced by one or more of the protein
subunits. Methanococcus jannaschii has an RNase P RNA that is not active in RNA
alone reactions, and it is missing its major substrate recognition regions. Apart from the
differences between the RNAs, these enzymes apparently have at least four proteins
similar to those of found in the yeast nuclear RNase P holoenzyme. A comparison of the
enzymatic properties of methanogenic archaeal RNase P enzymes will give more insight
into the ability of an RNase P to function without P8 or L15. Comparing both of them to
other RNase P enzymes will allow us to understand how bacterial-like RNAs interact
with eukuryotic-like protein subunits. Ultimately, the protein subunits of Methanococcus
jannaschii RNase P will allow us to further study the differences of these two enzymes
and to better understand the coevolution and exchange of function between RNA and
protein in a diverse ribonucleoprotein complex.
Materials and Methods
RNase P Activity Assays
RNase P assays were performed using
32
P-labeled pre-tRNAAsp from Bacillus subtilis
transcribed in vitro by run-off transcription of linearized (BstNI) pDW128 in the presence
of α32P GTP. The reaction conditions of all RNase P reactions were 50mM Tris (pH 8),
0.05% Nonidet P 40, 100mM ammonium acetate, 25mM magnesium chloride, and 2nM
(ca. 4000cpm) pre-tRNA, unless otherwise stated.
Assays were carried out at
temperatures ranging from 25-100°C. Enzyme was diluted such that percent cleavage
8
ranged in general from 20 to 70 percent of the total pre-tRNA added to the reaction. The
reaction products were then separated on 12% acrylamide 8M urea gels and visualized by
phosphorimagery.
Purification (Figure 3)
Cultivation of M. jannashii
Methanococcus jannaschii was grown in 20-ml starter cultures using ATCC media 1343
under 40psi of 60% H2:40% CO2 at 80°C. Starter cultures were grown for three days,
refreshing the gas daily. These
cultures (totaling 100ml) were injected in a New
Brunswick 13-L fermentor filled with the same medium. The fermentator was run at
80°C under one atmosphere of pressure (60%H/40%CO2) with the gas flow as low as
possible and reduced every 24 hours with 5 ml of 15% sodium sulfide. The fermentation
was run until the culture reached turbidity of about 0.6 OD600 (typically 2-4 days). The
cells were then harvested by centrifugation and frozen at -80°C.
Preparation of Cleared Lysate
Frozen cell pellets were ground with liquid nitrogen in a mortar and pestle. Cell powder
was then resuspended in 10ug/ml DNase I and TMGN-100 (50mM Tris, 10mM MgCl2,
and 100mM NH4Cl) at about 3ml per gram of starting material (27). The solution was
cleared at 16,000 X g for 30 min at 4°C. The supernatant was dialyzed overnight in two
exchanges of TMGN100 using Snakeskin dialysis membrane 3500 MWCO (Pierce).
9
Cleared lysed Cell Material
Dialysis into TM GN-100
DEAE sepharose column
Cs 2SO4 Gradient
Cs 2SO4 Gradient
Dialysis into TM GN-100
Q sepharose column
Glycerol Gradients
Affinity Purification
Cs 2SO4 Gradient
Figure 3. Flow chart of the RNase P purification from Methanocccus jannaschii
10
DEAE Sepharose Chromatography
200ml of DEAE sepharose was equilibrated in TMGN-100, fines were discarded and the
matrix was poured into a 4.5 x 44cm column and washed with 1.5L of TMGN-100 at
1.2ml/min. The 300mls of cleared dialyzed lysate were then loaded onto the DEAE
sepharose column at 0.5ml/min and washed with 1L of TMGN-100 at 1ml/min. The
column was eluted with a 1 liter linear gradient of 100mM to 500mM NH4Cl at
0.4ml/min followed by another 500mls of 500mM NH4Cl. Eighty-six 12ml fractions
were collected. Column fractions were assayed for RNase P activity and active fractions
were pooled and dialyzed with TMGN-100 overnight.
Cs2SO4 Buoyant Density Gradient Centrifugation
Cs2SO4 was added to the pooled active fractions from the DEAE column until a density
of 1.39g/ml was reached. The solution was added to 60ml quick-seal ultracentrifuge
tubes and centrifuged at 130,000 x G for 48 hours at 8°C. Fractions were collected from
the bottom of the tube in 1.2ml aliquots. The collected fractions were assayed for RNase
P activity and active fractions were pooled, sealed into new tubes, and centrifuged as
before. Active 0.5ml fractions were pooled and dialyzed into TMGN-100 for use on the
Q-sepharose column.
Q-Sepharose Chromatography
50ml of Q-sepharose were equilibrated in TMGN-100 and the fines were discarded. The
matrix was then poured into a 4.5 x 110cm column and 500mls of TMGN-100 were
11
passed through at 1ml/min. The 30mls of active RNase P from Cs2SO4 were loaded onto
the column at 1ml/min at 4°C. A 500ml wash was run through at 1ml/min. The RNase P
activity was eluted with a 500ml NH4Cl gradient from 100 to 1000mM in a total of 80 6.5
ml fractions. Fractions were assayed for activity and the peak fractions were further
purified through a glycerol gradient.
Glycerol Gradient Centrifugation
A 2ml glycerol gradient was made with TMGN-500, 0.025% NP40 and .02% SB12
Zwitterionic detergent.
The gradient was made in 1% steps ranging from 10-40%
glycerol. The gradient was made in a 3ml quickseal tube with the top cut off and
centrifuged at 95,000 x G in a Beckman TLS-55 swinging bucket rotor at 4°C. Fractions
(100ul) were then carefully removed from the top of the open tube and assayed for RNase
P activity. Each fraction (75ul) was then acetone precipitated (2 volumes acetone, frozen
overnight at -20°C) and, microcentrifuged for 20min, and resuspended in 20ul of protein
loading dye for SDS-PAGE.
Affinity Purification
Material from the Q sepharose column for affinity purification was dialyzed into binding
buffer (100mM NH4OAc, 50mM Tris-HCl pH 8, 0.01%NP40 and 25mM CaCl2)
Calcium chroride was used to limit the cleavage reaction. Biotin-labeled pre-tRNA was
synthesized by run-off transcription from linearized (BstNI) pDW128 in the presence of
0.5mM 11-biotin UTP (1:2 ratio with UTP) (Sigma) (32). Agarose-streptavidin beads
12
were blocked with 10mg/ml poly A, 25ug/ml glycogen, 20ug/ml BSA, 50mM Tris-HCl
(pH 8), 100mM NaCl, and 0.01% NP40 (32). The beads were washed twice with 50mM
Tris-HCl, 100mM NaCl and 0.01%NP40, followed by two additional washes with
binding buffer. To determine the ratio of biotin labeled pre-tRNA to column matrix
needed, pilot experiments using radiolabeled biotinalated pre-tRNA were prepared to
measure binding capacity. In general at least 10nmol biotin-labeled pre-tRNA was added
to the enzyme preparation (500ul) and allowed to bind for one minute. The mixture was
then added to the 400ul of pre-blocked beads. Beads were then washed three times by
pelleting in a microfuge for one minute, the supernatant was discarded, and an equal
volume of binding buffer was added. The beads were then washed twice with RNase P
buffer. RNase P was eluted from the beads with RNase P reaction buffer containing
10nmol pre-tRNA (Figure 4). In order to remove the final remnants of activity from the
beads they were placed in 1.39g/ml Cs2SO4 gradients in TMGN-100, and centrifuged as
described above.
Protein Gels and Quantification
Protein concentrations were determined by BCA kit (Pierce) or from silver stain gels and
known molecular weight markers (Sigma). Quantification of protein by silver staining
was carried out by making a series of dilutions of a BCA stock and running them next to
13
Pre-bound enzy me
Supernatent after binding
Binding buffe r wash
P assay buffer wash
P assay buffer wash
P assay buffer with cold p re-t RNA
P assay buffer with cold p re-t RNA
Beads after elution
1.0
0.75
0.5
0.25
0
Negative Control
Fraction Cleaved
Figure 4. Affinity column results graphed as Fraction Cleavaged. Assays were done in
30mM Mg2Cl and 100mM NH2Cl for 1min at room temperature.
14
Northern Blots
Gels were 8% acrylamide and 8M urea with a 4% stacking gel, using RNA loading dye
run at 300V until the bromphenol blue dye reached the bottom. RNA standards were
purified and quantitated in vitro transcripts of the Methanococcus jannaschi RNase P
RNA (used in both gels and slot blots). The standard was quantified by ethidium
bromide spot testing and in Ribogreen assays (Molecular Probes). The standard was
loaded in decreasing amounts from 1000-50ugs. The gel was then electroblotted for two
hours on to Hybond-N filter (Amarsham). Once the filter was transferred it was UV
cross-linked and placed in a 100ml hybridization bottle for prehybridization. Slot blots
were baked at 80°C for 2hr before cross-linking. The prehybridization was done in 1X
Denhart’s solution, 20 mM Na2HPO4 pH 6.5, 1mM DTT, 5X SSC and 100ug/ml of poly
A. After two hours at 65°C in vitro transcribed Methanococcus jannaschi RNase P
antisense probe RNA was added to the hybridization bottle and allowed to hybridize at
65°C overnight. Filters were washed twice with 2X SSC and 0.1% SDS for 5 minutes
each at room temperature, followed by two additional washes of 1X SSC and 0.1% SDS
for 20 minutes at 65°C. Filters were then visualized by phosphorimagery.
Concentration of RNase P Samples
The concentration of RNase P activity was carried out mainly by placing the sample in
Snakeskin (Pierce) dialysis tubing and pouring PEG 8000 over the dialysis bag in a small
pyrex dish, changing the PEG frequently. Once the desired volume of sample was
reached, the bag was removed, washed with deionized water, reclipped tightly and
dialyzed in the buffer of choice.
15
Amicon Micron tubes (10kDa cut off) were also used for concentration as instructed by
the company. After using the Micron tubes in early parts of the experiments it was
noticed that the activity was not increasing proportionally with concentration. Further
testing revealed that RNase P activity was selectively passing through the membrane,
although the filters efficiently retain BSA.
Size Exclusion
A size exclusion column was run to determine the approximate size of the holoenzyme.
Twenty milliliters sepharose CL-4B was equilibrated and poured in a 0.8 x 25cm column.
Protein markers (Sigma) ranging from 29,000 to 700,00 were prepared as instructed.
Markers were mixed with a partially-purified RNase P sample, and 100ul were loaded
into the column, which was run at 3ml/hour. Fractions were assayed for activity and the
absorbance at 280nm was measured to identify markers.
Results
Purification
Approximately 105 grams (wet weight) of cell material was used for the purification.
After being lysed, cleared and dialyzed, the material was run through DEAE-sepharose
and eluted at ca. 350mM to 400mM NH4Cl (Figure 5). The active fractions numbering
25-45 were pooled for the next step. After dialysis these pooled fractions were subjected
16
to Cs2SO4 gradient centrifugation. Activity peaked in the middle fraction of the gradient,
but the entire middle third of the gradient contained enzyme activty. This portion of the
gradient was diluted and adjusted to a density of 1.39 g/ml and recentrifuged (Figure 6).
The peak activity of both gradients was found to be at a buoyant density of 1.39 g/ml.
The active fractions from the second gradient were dialyzed for Q-sepharose
chromatrography. RNase P activity eluted from the Q-sepharose column at ca. 650mM
NH2Cl (Figure 7). Northern hybridization from the peak active fraction (44) indicate an
RNase P RNA concentration of ca. 400ug/ml. This fraction had bands that would appear
faintly on a Coommassie stained gel however, the amount of background was still too
high to evaluate which bands were copurifying with RNase P activity. A glycerol
gradient of a portion of the most active Q-sepharose fraction showed six bands on a silver
stained 12% SDS-PAGE gel that co purify with RNase P activity. The apparent
molecular weights of the bands were 83.1 KDa, 65.6 KDa, 48.2 KDa, 19.9 KDa, and 16.8
KDa. The 16.8Kd band appears to be a doublet. The bands present in the peak activity
of the glycerol gradient were also seen in the affinity column fractions of peak activity.
Bands visible on a 12% silver stained SDS-PAGE gel were comparable in size to those
seen in the active fractions of the glycerol gradients and the affinity column. Six bands
were identified and their molecular weights are 16.7 (1), 28.2 (4), 48.2 (5), 65.6 (6), and
83.1 KDa (7) (Figure 8). Peak samples from
17
0.5
0.3
Pooled Fractions
Fraction cleaved
0.2
0.1
84
81
78
75
72
69
66
63
60
57
54
51
48
45
42
39
36
33
30
27
24
21
18
15
12
9
0
3
Fraction cleaved
0.4
fraction
Figure 5. DEAE-Sepharose chromatrography of Methanococcus jannaschii cleared
lysate. The fraction of pre-tRNA cleaved versus fractions along a 100-500mM NH2Cl
gradient. All fractions were 12ml with the peak at ca. 250-350NH2Cl.
18
80
0.5
0.8
% Clevage
0.4
60
0.6
Abs 280nm
0.3
40
0.2
0.2
20
0.1
0.1
1.33
1.32 4
1.324
1.33 3
1.356
1.38 4
1.406
1.40 1
1.426
1.42 8
1.442
1.44
1.49
1.48 5
1.50 1
1.539
1.56 2
0
1.63
0
Figure 6. First round Cs2SO4 gradient fractions were removed from the bottom and the
density (g/ml), fraction cleaved, and absorbance at 280nM was measured with the peak of
absorbance occuring at the density of ribosomes.
19
80
Fraction used
for Glycerol
gradients
Fraction Cleaved
60
Fraction Cleaved
40
20
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
48
50
52
54
56
58
60
62
64
0
Fraction
Figure 7. Q sepharose chromatrography. Fraction of pre-tRNA cleaved verses fractions
collected along a 100-1000mM NH4Cl gradient with the peak eluting at ca. 600700NH2Cl.
20
Fraction Cleaved
Pooled Fractions
0.6
Bottom
0.4
0.2
Top
0
1
3
5
7
9
11
13
15
17
Fraction
8
7
6
5
4
3
2
1
Figure 8. RNase P activity in glycerol gradients over a SDS-PAGE gel of the same
fractions. Bands purifying with RNase P are numbered to the right of the band 1-8.
21
the glycerol gradient (7-10) were then sent to Denise Meagher at the Protein/DNA
technology center of Rockefeller University for identification by trypsin digest, mass
spectrometry, and comparison with the complete genomic sequence of Methanococcus
jannaschii.
The Effects of Monovalent and Divalent Salts on RNase P Activty
RNase P assays were performed with varying amounts of MgCl2 to determine peak
activity (Figure 9). The broad peak of activity is centered around 30mM MgCl2, but
there is only a 10% difference in activity between 20-50mM MgCl2. Never the less
MgCl2 is required for activity, this is evident by the fact that the addition of 1nM EDTA
will eliminate RNase P activity. MgCl2 can be diluted 1:35 in the presence of CaCl2,
reducing the amount of total activity to almost zero. This can then be recovered by
adding MgCl2 back into the solution, returning activity to its normal levels (Figure 10).
Monovalent salt titration was tested using NH4OAc ranging from 20mM to 1500mM in
standard RNase P assay conditions. There is approximately 70 to 80 percent cleavage
between 20mM and 500mM NH2OAc (Figure 11).
22
0.4
Fraction cleaved
Fraction Cleaved
0.3
0.2
0.1
100
80
60
50
30.2
20.2
12.2
10.2
8.2
3.2
1.2
0
MgCl2 (mM)
Figure 9. Fraction cleavaged of pre-tRNA as a function of MgCl2 concentration. Assays
were done at 60°C for 2 minutes.
23
350uM MgCl2 and 10mM CaCl2
5
0°C 22°C
30
0°C 22°C
60
700uM CaCl2 and 10mM MgCl2
5
30
0°C 22°C 0°C 22°C 0°C 22°C
60
0°C 22°C
Starting Material with 10mM MgCl2
5
0°C
30
22°C 0°C
22°C
60
0°C 22°C
Assays were done at 22°C or 0°C
Figure 10. Enzyme was originally stored in TMGN-20 which was exchanged for TCG
(same as TMG except the MgCl2 was replaced with CaCl2) in microcon (Amicon)
centrifuge tubes. MgCl2 was diluted from 10mM to 350uM while CaCl2 was brought up
to 10mM. A sample from this was removed and the reverse approach was applied taking
CaCl2 to 700uM and bringing MgCl2 back to 10mM. Activity for the starting material
and both dilutions was tested at 5, 30 and 60 min. on the bench and on ice.
24
NH4OAc (mM)
Figure 11. Fraction of pre-tRNA cleaved as a function of NH4OAc concentration.
Assays were done at 60°C for 2 minutes.
25
The Effects of Temperature on RNase P Activity
RNase P from Methanococcus jannaschii is highly thermal stable, as would be expected
from an organism that grows at 80°C. The RNase P activity increases with temperature
up to 90°C, above which point the activity is impossible to measure due to the extent and
speed at which nonspecific hydrolysis of the substrate occurs (Figure 12).
Enzyme Kinetics
The enzymatic properties of the Methanococcus jannaschii RNase P were evaluated by
reaction velocity as a function of pre-tRNA concentration. The Km was found to be
68nM and the Kcat was determined to be 34nM per minute. The kinetic properties of M.
jannaschii RNase P are similar to other characterized RNase P holoenzymes and
particularly that of Methanobacterium thermoautotrophicum (Km of 37nM, Kcat
unknown) (Figure 13).
Discussion
Purification of the Methanococcus jannaschii RNase P holoenzyme
The crucial step in the purification of this enzyme is the Cs2SO4 gradients. In a largescale preparation it would not be a plausible first step due to the amount of material that
would have to be made into gradients. DEAE-sepharose chromatrography removes large
amounts of protein, but apparently does not remove tRNAs and ribosomes.
Unfortunately, tRNA in the sample inhibits the enzyme competitively. Ribosomes pose
the biggest problem in the purification of RNase P, due to the large amount of ribosomal
26
1
350000
Fraction Cleaved
total counts
300000
250000
0.8
200000
total counts
Fraction Cleaved
0.9
0.7
150000
80
60
40
100000
20
0.6
Temp C
Figure 12. Fraction of pre-tRNA cleaved as a function of temperature while pre-tRNA
degradation is expressed as a loss of total counts. Assays were done for one minute and
put back on ice. RNase P activity increases with temperature past the melting point of
pre-tRNAAsp (ca. 65°C).
27
8
7
1/nM mature tRNA
6
5
4
3
2
1
0
0
0.1
0.2
0.3
0.4
0.5
0.6
1/nM pre-tRNA
Figure 13. Lineweaver-Burk plot. The result from RNase P activity relative to the
concentration of substrate. The Km is 68nM and the Kcat is 37nM min-1.
28
material present in the cell compared to RNase P and the similar biochemical properties
of RNase P ribosomes. However, the Cs2SO4 gradients efficiently separate both the
ribosomes (that have a buoyant density of 1.45g/ml) and tRNA (that has a buoyant
density with the bulk nucleic acid of 1.6g/ml) from RNase P (that has a buoyant density
of 1.39g/ml).
Other column matrices were tried without success; both CM sepharose and heparin
cellulose failed to provide any purification. In fact no activity could be eluted from
either. However, the combination DEAE ion exchange, two rounds of Cs2SO4 buoyant
density, and back to Q sepharose ion exchange, worked well even compared to pilot
experiments using density gradients initially to remove the ribosomes and inhibitors.
Pilot experiments using sucrose gradients after all previous steps, gave little if any
purification. The material following glycerol gradient centrifugation was clean enough
for protein identification, thereby removing the need for affinity purification.
Affinity Purification of RNase P
Affinity purification is generally the most selective way to purify enzymes of interest. In
the case of RNase P premature cleavage must be prevented until unbound material can be
removed. Other RNase P affinity purifications have been published; one use biotin prebound to the beads and the other uses sulfalink and GMPS-labeled pre-tRNAs (33,34).
Neither of these methods proved successful with the M.jannaschii enzyme. The
procedure developed for the affinity column described here works, but has two
drawbacks; (1) only a small amount of material can be purified at a time, (2) eluting
29
RNase P activity off of the beads is difficult. Attempts at larger scale affinity columns
(15ml) were not as effective as those done at a smaller scale (1.5 ml tubes). A much
smaller amount of total activity could be bound to the beads when the procedure was
scaled up. The use of smaller volumes meant that the samples would have to be
subsequently concentrated, and multiple rounds of purification were needed. After
material was bound to the column the only way to remove a majority of it was by Cs2SO4
gradient centrifugation. This was successful for eluting and separating the beads from
RNase P, but it may also disrupt a fraction of the enzyme (judging from silver gels of
both affinity and glycerol purification). Given a more efficient and feasible alternative
(glycerol gradients), this affinity procedures was not used.
Fold Purification
The Q-sepharose column has an estimated fold purification of ca. 600 fold (Table 1).
However, we believe this to be an underestimate of the actual purification (judging from
the SDS-PAGE gels). The glycerol gradient purified material was estimated at having a
purity of a hundred fold greater the Q sepharose column. However, methods for
determining protein concentration are usually either based on a few specific types of
amino acids or they rely on the fact that all of the proteins in the sample will show up on
a gel, and no two proteins are the same molecular weight.
Evaluating the Size of Methanococcus jannaschii RNase P
The Methanococcus jannaschii RNase P has an apparent density in Cs2SO4 (1.39g/ml)
between the density for protein alone and RNA alone and closer to the density of the
30
Fraction
units
protein (mg/ml)
Units/mg
fold purity
cleared cell lysisate
15136
21
717.9
1
DEAE
26662
6.2
4270.6
5.9
Cs2S04
103210
3.2
32530.7
45.3
Q sepharose
355740
0.76
467605.2
651.3
Unit = nM cleaved per 1 ml of sample
Table 1. Fold purification estimated using BCA method for protein quantification and
RNase P activity as described.
31
eukaryotic enzyme than to that of Bacteria (24). This is consistent with the density of
Methanobacterium thermoautotrophicum (1.42 g/ml) (personal communication T. Hall).
If it assumed that the density is directly related to the ratio of RNA to protein, then
Methanococcus jannaschii RNase P holoenzyme is ca. 73% protein and ca. 27% nucleic
acid. The RNA component should be approximately 79kDa (260nt) the protein
component is approximately 213kDa. This is certainly an optimistic simplification
however, the lower density of RNase P does seem to correlate with the greater need for
protein in the RNase P holoenzyme (24). The molecular weight of the RNase P
holoenzyme appears to be approximately 400kDa, estimated using protein molecular
markers in size exclusion chromatography and the total molecular weight of the RNA and
proteins that apparently copurify with enzyme activity is about 300kDa (this is not
accounting for band 1 that may be two proteins (figure 8)). Size exclusion
chromatography, as well as the additive estimated molecular weight of the bands
appearing on purified RNase P gels agrees with the large size of RNase P in
Methanococcus jannaschii.
Enzymatic Differences between the Methanogenic Archaeal RNase P Holoenzymes
The differences in the catalytic parameters of the RNase P of methanogenic Archaea are
evident in the holoenzyme as well as the RNA alone reaction. The M.
thermoautotrophicum RNase P RNA alone is active (in high ionic conditions) whereas
the RNA from Methanococcus jannaschii is not (25). While the holoenzymes of both
RNase Ps are active, the M. thermoautotrophicum RNase P (personal communication T.
Hall) has a narrow range of optimal activity (800mM NH4OAc and 5-10mM Mg2Cl) but
32
the M. jannaschii RNase P holoenzyme has a wide optimal range of salt conditions (20500mM NH4OAc and 1-100mM MgCl2). M. jannaschii RNase P has a buoyant density
of 1.39g/ml and a Km of 68 nM whereas M.thermoautothrophicum RNase P has a
buoyant density of 1.42g/ml (closer to that of ribosomes at 1.45g/ml) and a Km of 37nM.
It is not clear what role the changes in RNA structure play in these differences.
Proteins Purifying with Methanococcus jannaschii RNase P Holoenzyme
Six potential RNase P proteins were identified on the basis of copurification with activity
on the glycerol gradient; their molecular weights were 16.7 (band 1), 28.2 (band 4), 48.2
(band 5), 65.6 (band 6), and 83.1 kDa (band 7) (Table 2). Three of these bands are
consistent with the size of open reading frames in the M. jannaschii genome homologous
to Methanobacterium thermoautotrophicum RNase P proteins. Band 1 has molecular
weight that is consistent with M. jannaschii open reading frames MJ0494 and MJ0962.
MJ0494 is similar to a protein in the yeast holoenzyme (Pop5p) and M.
thermoautotrophicum MTH687 (Figure 14). MJ0962 is similar to M.
thermoautotrophicum MTH1618 and Rpr2p from yeast (Figure 15). Band 4 has the
correct molecular weight for the open reading frame MJ1139 that is similar to Rpp1p and
MTH688 (Figure 16). A fourth open reading frame in M. jannaschii, MJ0464, is too
small to be seen on this gel. This protein corresponds with MTH11 and Pop4p (Figure
17). These proteins were first identified in the purification of the Saccharomyces
cerevisiae RNase P holoenzyme, and then after a careful genomic search they were found
in the Methanobacterium thermoautotrophicum (MTH11, MTH1618, MTH687, TH888)
(21) (personal communication with T. Hall). Two of these proteins are of similar
33
Pop5p (yeast)
MTH687 (M.thermo.)
MJ0494 (M. jannaschii)
PH1481 (P. horikoshii)
Pf1293409 (P. furiosus)
PAB0467 (P. abyssii)
AF0489 (A. fulgidus)
1
1
1
1
1
1
1
------------MVRLKSRYILFEIIFPPTDTNVEESVSKADILLSHHRASPADVSIKSI
-----------------------MKILPPTLR-------VPRRYIAFEVISERELSREEL
-------------------MIEMLKTLPPTLR-------EKKRYIAFKILYDEELKEGEV
-------------------MMRKLKTLPPTLR-------DKNRYIAFEIISDGDFTKDEV
-------------------MSERPKTLPPTLR-------DKKRYIAFKVISENQFNKDEI
--------------------MMKLKTLPPTLR-------DKNRYIAFEIISDDEFTKDEV
MHGSRGNFRNHHKLFFTFRSKAIVKGLPPSLR-------SRKRYIAFRIIAEKKIDERSL
48
30
34
34
34
33
53
Pop5p (yeast)
MTH687 (M.thermo.)
MJ0494 (M. jannaschii)
PH1481 (P. horikoshii)
Pf1293409 (P. furiosus)
PAB0467 (P. abyssii)
AF0489 (A. fulgidus)
49
31
35
35
35
34
54
LQEIRRSLSLNLGDYGSAKCNSLLQLKYFSNKTS------TGIIRCHREDCDLVIMALML
VSLIWDSCLKLHGECETSNFR--LWLMKLWRFDFPDAVRVRGILQCQRGYERRVMMALTC
VNLIRKAVLEYYGSWGTSKAN--PWLVYYDFP--------YGILRCQRDNVDYVKASLIL
KELIWKSSLEVLGETGTAIVK--PWLIKFDPNTK------TGIVRCDREYVEYLRFALML
KEAIWNACLRTLGELGTAKAK--PWLIKFDETTQ------TGIIRCDRNHVYDVIFSLTL
KSLIWEASLRVLGELGTALAK--PWFIKYDPKTK------TGIVRCDREYVEHLRFALML
SRALSEKMLSLFGECFAASG---LRLEAFDGE--------RGIVRCYREALDKVMVALTL
102
88
84
86
86
85
102
Pop5p (yeast)
MTH687 (M.thermo.)
MJ0494 (M. jannaschii)
PH1481 (P. horikoshii)
Pf1293409 (P. furiosus)
PAB0467 (P. abyssii)
AF0489 (A. fulgidus)
103
89
85
87
87
86
103
MSKIGDVDGLIVNPVKVSGTIKKIEQFAMRRNSKILNIIKCSQSSHLSDNDFIINDFKKI
AHHHSGVR-VAIHILGLSGTIRSATQKFIKPSKKDK--------------------Y--IREFKEKP-VNIICLGVSGTIRKAKIKFLGIKKPKR--------------------WFVI
VSEFNGKR-LIIRTLGVSGTIKRLKRKFLAKYG-----------------------WK-VSDINGNK-AIIKVLGVSGTIKRLKRKFLSQFG-----------------------WR-ATDFNGKR-LIIRTLGVSGTIKRLKKKFLSQYG-----------------------WK-MTHVGGVR-VIPLTLGVSGTIKRCKRKYLEV-----------------------------
162
124
123
120
120
119
132
Pop5p (yeast)
MTH687 (M.thermo.)
MJ0494 (M. jannaschii)
PH1481 (P. horikoshii)
Pf1293409 (P. furiosus)
PAB0467 (P. abyssii)
AF0489 (A. fulgidus)
163
124
124
120
120
119
132
GRENENENEDD
----------RRERLKAKKQK
-----------------------------------------
173
124
134
120
120
119
132
Figure 14. Alignment of possible archaeal homologs of the Yeast RNase P subunit
Pop5p. Residues are highlighted with a 60% shading threshold in black (identical) and
gray (similar) using the BLOIUMB2 matrix.
34
Rpr2p (yeast)
MTH1618 (M. thermo.)
MJ0962 (M. jannaschii)
AF0109 (A. fulgidus)
PH1601 (P. horikoshii)
PAB0385 (P. abyssi)
1
1
1
1
1
1
MGKKAHGGKMKPEIDENGTLLVPPPRTIANQDHFHRLNYLYQISAYQTRARQKARTDAHT
---------MRRGKRPRWMLKIAEER----------------IDILFRMADREFSAN--P
----------MKKFLEKKLKKIAYER----------------IDILMSLAEEEAKKGN-W
--------MLRRD--KKRESRIARER----------------VFYLIKRAEEWKNID--Y
----MVDIVKRRDWEKKEKKKIAIER----------------IDTLFTLAERVARYS--P
--------MKKVSWEKREKKKVAIER----------------IDTLFTLAEKVVKYS--P
6
3
3
3
3
3
Rpr2p (yeast)
MTH1618 (M. thermo.)
MJ0962 (M. jannaschii)
AF0109 (A. fulgidus)
PH1601 (P. horikoshii)
PAB0385 (P. abyssi)
61
34
34
33
39
35
PLARNYIKSMDLISKKTKTSLLPTIKRTICKKCHRLLWTPK--KLEITSDG--ALSVMCG
HRSHRYTELARNIAMKYRVRIPREWRRRFCRKCYSFLKPGANCTVRIADG---KVNFRCH
DRAKRYVYLARRIAMKMRIRFPKKWKRRICKKCGTFLLYGRNARVRIKSKRYPHVVITCL
ELARRYVELARKIAMRYRVRIPRELKATYCKKCLYPYKAGK-FRVRVRKSR---VIITCL
DLAKRYVELALEIQKKAKVKIPRKWKRRYCKRCHTFLIPGVNARVRLRTKRMPHVVITCL
DLARRYVELALEIQKKSKVKLPRKWKRRYCKRCHAFLVPGFNARVRLRTDRMPHVVITCL
1
9
9
8
9
9
Rpr2p (yeast)
MTH1618 (M. thermo.)
MJ0962 (M. jannaschii)
AF0109 (A. fulgidus)
PH1601 (P. horikoshii)
PAB0385 (P. abyssi)
116
91
94
89
99
95
-CGTVKRFN-IGADPNYRTYS--ER---EGNLLNS--------------ECGHIMRFPYIREKKDRRRNKIESHTTKEGTDEQITVGAHNKCGESQSDR
ECGAIYRIPMIREKKEKRRKKLEERLKAKSNSQTS--------------NCGFERRIP-IRPKRVNRKV-----------------------------ECGYIMRYPYLREVKQKRKKAT---------------------------ECGHIMRYPYLREVKEKRKRKKD---------------------------
144
140
128
107
120
117
Figure 15. Alignment of possible archaeal homologs of the Yeast RNase P subunit
Rpr2p. Residues are highlighted with a 60% shading threshold in black (identical) and
gray (similar) using the BLOIUMB2 matrix.
35
Rpp1p (yeast)
p30 (human)
MTH688 (M. thermo.)
MJ1139 (M. jannaschii)
AF2317 (A. fulgidus)
PH1877 (P. horikoshii)
1
1
1
1
1
1
---MLVDLNVPWPQNS--YADKVTSQAVNNLIKTLSTLHMLGYTHIAINFTVNHSEKFPN
---MAVFADLDLRAGS----DLKALRGL------VETAAHLGYSVVAINHIVDFKEKKQE
MIPQRILMKFFDFHIQGRDHDS-SLRLL------LE-ASRLGYQGGVLVYPSERYPD-----MRIDINRIEKEE-----D---IKLL------KE-LKWNGFV--FYQYDDEFSKD-----MYDFLRFFPENLP----D-------------------LGFK--SYVFMLE-------MVGGGGVKFIEMDIR----D---K-EA------YE-LAKEWFD--EVVVSIKFNEE---
55
47
49
37
25
39
Rpp1p (yeast)
p30 (human)
MTH688 (M. thermo.)
MJ1139 (M. jannaschii)
AF2317 (A. fulgidus)
PH1877 (P. horikoshii)
55
48
49
37
25
39
-DVKLLNPIDIKRRFGELMDRTGLKLYSRITLIIDDP----SKGQSLSKISQAFDIVAAL
IEKPVAVSELFTTLPIVQGKSRPIKILTRLTIIVSDPSHCNVLRATSSRAR-LYDVVAVF
------LKSDLESLRENP-ELQDFEIARGVMINASDP---RDMRRSVNKFRKKADVIYVS
------R---YEEVKAIA-ESYKLKVYSGVKIKTESS---KQLRDKVKKFRNKCHIILIE
-------------------KPKG----CGIVIRANSP---EELRKKLRGV--KRRAIVGI
------VD--KEKLREAR-KEYG---KVAILLSNPKP---SLVRDTVQKF--KSYLIYVE
110
106
99
84
57
82
Rpp1p (yeast)
p30 (human)
MTH688 (M. thermo.)
MJ1139 (M. jannaschii)
AF2317 (A. fulgidus)
PH1877 (P. horikoshii)
111
107
100
85
58
83
PISEKGLTLSTTNLDIDLLTFQYGSRLPTFLKHKSICSCVNRGVKLEIVYGYALR-DVQA
PKTEKLFHIACTHLDVDLVCITVTEKLPFYFKRPPINVAIDRGLAFELVYSPAIK-DSTM
GGNLKVNRAACESRRVDVLSAPYTSRRDPGINHVLAREAARNNVAVELPLADVIGSWLKV
GGVLKINRAAVELHDVDILSTPELGRKDSGIDHVLARLASNHRVAIELNFKTLLNKDGYE
IGKEAVCREAVMRRRVDVILDWED-RE---LDYATLKLAAEKDVAIELSLSKFLRTEGYK
SNDLRVIRYSIEKG-VDAIISPWVNRKDPGIDHVLAKLMVKKNVALGFSLRPLLYSNPYE
169
165
159
144
113
141
Rpp1p (yeast)
p30 (human)
MTH688 (M. thermo.)
MJ1139 (M. jannaschii)
AF2317 (A. fulgidus)
PH1877 (P. horikoshii)
170
166
160
145
114
142
RRQFVSNVRSVIRSSRSRGI--VIGSGAMSPLECRNILGVTSLIKNLGLPSDRCSKAMGD
RRYTISSALNLMQICKGKNV--IISSAAERPLEIRGPYDVANLGLLFGLSESDAKAAVST
RARVLEQFREILKLHRKFGFPLLLTSRASSIYDLRTPGDIMNLAECFGMESSEAEESLTS
RARTLLFFRNNLKLAKKFDVPVVISTDAENKYQIKNPYDLRAFLNTLVEPLYAKKIMETA
RMHLFERLRQEIMVIRKFDVPFIVTTAAENQYELRTRKQVETFFKFFGAEIPKARLYAQR
RANLLRFMMKAWKLVEKYKVRRFLTSSAQEKWDVRYPRDLISLGVVIGMEIPQAKASISM
227
223
219
204
173
201
Rpp1p (yeast)
p30 (human)
MTH688 (M. thermo.)
MJ1139 (M. jannaschii)
AF2317 (A. fulgidus)
PH1877 (P. horikoshii)
228
224
220
205
174
202
LASLVLLNGRLRNKSHKQTIVTGGGSGNGDDVVNDVQGIDDVQTIKVVKRSMDAEQLGHA
NCRAALLHGETRKT---AFGIISTVKK----------PRPSEGDEDCLPASKKAKCEG-TPASILEDSGNR-----HLLIAEGVR--------------------LLPES--------YKICDFRDYLMR-----DNVVRYGVE--------------------IIKEEKE------LVRRYFD----------ENYIMDGFE--------------------VEQLSNSGVV---YPEIILK----R-----LKY----------------------------------------
287
268
245
232
199
212
Figure 16. Alignment of possible archaeal homologs of the Yeast RNase P subunit
Rpp1p. Residues are highlighted with a 60% shading threshold in black (identical) and
gray (similar) using the BLOIUMB2 matrix.
36
APE0362 (A. pernix)
Pop4p (yeast)
Pop4p (human)
C15C6.4. (C. elegans)
MTH11 (M. thermo.)
MJ0464 (M. jannaschii)
AF1917 (A. fulgidus)
PAB2126 (P. abyssi)
PH1771 (P. horikoshii)
1
1
1
1
1
1
1
1
1
-----------------------------------------------------------MDRTQTFIKDCLFTKCLEDPEKPFNENRFQDTLLLLPTDGGLTSRLQRQQRKSKLNLDNL
-------MKSVIYHALSQKEANDSDVQPSGAQRAEAFVRAFLKRSTPRMSPQA--REDQL
-----------MSKDVRIGEPGESTSRGGGDRLGVLHLEKNVTSHFKRKSKKT--EKTNL
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~MRRNGKERKDRTS~~GGSQR
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~MRRNSKERKNRAT~~RRSQG
1
60
51
47
1
1
1
18
18
APE0362 (A. pernix)
Pop4p (yeast)
Pop4p (human)
C15C6.4. (C. elegans)
MTH11 (M. thermo.)
MJ0464 (M. jannaschii)
AF1917 (A. fulgidus)
PAB2126 (P. abyssi)
PH1771 (P. horikoshii)
1
61
52
48
1
1
1
19
19
--------------------------MKGRR----------------------------QKVSQLESADKQLEKRDYQRINKNSKIALREYINNCKKNTKKCLKLAYENKITDKEDLLH
QRKAVVLEYFTRHKRKEKKKKAKGLSARQRRELRLFDIKPEQQRYSLFLPLHELWK---TRGRLTEDDKKAFKFADIVPMNTDWQKYFRERLG------------------------------------------------------M----------------------------------------------------------M-------------------------------------------------------MRGR-----------------------------PYQEIVGRTWIFRG~~~~~~~~~~~SHRGR~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
SYQEIIGRTWIFRG~~~~~~~~~~~AHRGR~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
5
120
107
81
1
1
4
37
37
APE0362 (A. pernix)
Pop4p (yeast)
Pop4p (human)
C15C6.4. (C. elegans)
MTH11 (M. thermo.)
MJ0464 (M. jannaschii)
AF1917 (A. fulgidus)
PAB2126 (P. abyssi)
PH1771 (P. horikoshii)
5
121
107
81
1
1
4
37
37
----------------------------------------------PSHRRQLIPPLLTG
YIEEKHPTIYESLPQYVDFVPMYKELWINYIKELLNITKNLKTFNGSLALLKLSMADYNG
------------------------------QYIRDLCSGLKPDTQPQMIQAKLLKADLHG
-----------------------------------------NHFSSTKIEKTILKSDYHG
-----------------------------------------------ITPRNIFRHELIG
-----------------------------------------------ITPHNILRHELIG
-----------------------------------------------LQGVELIARDWIG
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~VTKRNIIWHELIG
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~VTRRNIIWHELIG
19
180
137
100
14
14
17
50
50
APE0362 (A. pernix)
Pop4p (yeast)
Pop4p (human)
C15C6.4. (C. elegans)
MTH11 (M. thermo.)
MJ0464 (M. jannaschii)
AF1917 (A. fulgidus)
PAB2126 (P. abyssi)
PH1771 (P. horikoshii)
20
181
138
101
15
15
18
51
51
LRVKVLAHSDPSLEGLEGWVVVEEARSLRILTLE---GRVSTVLKDLAVIEVEAPG---ALLRVTKSKNKTLIGLQGIVIWDSQKFFIMIVKGNIIDEIKCIPKKGTVFQFEIPISDDD
AIISVTKSKCPSYVGITGILLQETKHIFKIITKE---DRLKVIPKLNCVFTVETD----ALLTIWLAENPTQIGISGIVVLETRHTFQMVTQE---DRFVVIPKKGSVFRFILG----LSVRIARSVHRDIQGISGRVVDETRNTLRIEMDD---GREITVPKGIAVFHFRTPQ---LKVEIVEAKNKAMIGIKGKVVDETRNTLVIEKED---GREVVIPKDIAVFLFQLK----LMVEVVESPNHSEVGIKGEVVDETQNTLKIMTEK----GLKVVAKRGRTFRVWYK----LKVRVVNSMHPGFVGIEGYVVDETRNMLVIVGD~~~~~KVWKVPKDVCIFEFETED~~~~
LRVRIVGSTHPAFVGIEGYVIDETRNMLVIAGD~~~~~RIWKVPKDVCIFEFEADD~~~~
72
240
189
152
67
66
68
101
101
APE0362 (A. pernix)
Pop4p (yeast)
Pop4p (human)
C15C6.4. (C. elegans)
MTH11 (M. thermo.)
MJ0464 (M. jannaschii)
AF1917 (A. fulgidus)
PAB2126 (P. abyssi)
PH1771 (P. horikoshii)
72
241
189
152
67
66
68
101
101
--GEYIRISGRVLIGNPLDRVKEYRWRVSRRCRSSSRLKT-------DSALRYSILGDRFKYRSVDRAGR-KFKSRRCDDMLYYIQN---------GFISYIYGSKFQLRSSERSAK-KFKAKGTIDL---------------DRLFSLFGDGMRTRPAWRGKKPRIKRLLPTFIRGTLQTVPTVSTKD
--GELVEIDGRALVARPEERIKK-KFRKP--------------------GCKVKVDGRLLIGRPEERLKK-KIKILYPY-----------------GKIMRIKGDLINFRPEDRIKR-GLMMLKRAKGVWI----------~~GAKIKIPGERLVGRPEMRLKK~RWRKW~~~~~~~~~~~~~~~~~~~
~~GTKIKIPGERLVGRPEMRLKK~RWKKW~~~~~~~~~~~~~~~~~~~
110
279
220
198
93
95
102
127
127
Figure 17. Alignment of possible archaeal homologs of the Yeast RNase P subunit
Pop4p. Residues are highlighted with a 60% shading threshold in black (identical) and
gray (similar) using the BLOIUMB2 matrix.
37
Band
Number
1
2
3
4
5
6
7
8
Molecular
Weight (KDa)
17
20
22
28
48
66
83
120
Copurifi
cation
yes
maybe
maybe
yes
yes
yes
yes
maybe
Possible ORF
MJ0494, MJ0962
MJ1139
Table 2. Estimated molecular weight of bands appearing to copurify with RNase P on the
glycerol gradient gel.
38
molecular weights, approximately the size of band number 1, which appears to be twice
as intense as the other bands. Three other areas on the gel might possibly contain bands
copurifying with activity, and are numbered 2, 4, and 8. The molecular weights of these
bands are approximately 19.9, 21.5 and 119.4 kDa. Band two seems to have peaked
higher in the glycerol gradient than RNase P activity.
Future Research Areas
Once the proteins that apparently copurify with RNase P activity are identified by tryptic
digestion and mass spectroscopy (Protein/DNA technology center of Rockefeller
University) antiserum against identified open reading frames will be generated and their
relationships with RNase P tested via Western blots (testing copurification) and
immunoprecipitation (via depletion of activity). Any protein that does not have an
apparent homolog in A-type archaeal RNase P enzymes is a candidate protein that may
compensate for the loss of P8 and L15. This would be tested directly using 32P-labeled
5′end-azidophenacyl tRNA, circularly permuted to place the cross-linking agent into the
T-loop or CCA tail, to crosslink to protein and label proteins in the vicinity of these
recognition sites. The RNase P proteins from Methanococcus jannaschii similar to those
of Saccharomyces cerevisiae will be tested for the ability to complement yeast
knockouts.
The RNase P from Methanococcus jannaschii has been purified for the enzymatic
characterization and identification of protein subunits. Although the RNA subunit of the
enzyme lacks structures used in other RNase P RNAs for substrate recognition, the
39
properties of the holoenzyme are similar to those of other characterized RNase P
enzymes. It seems likely that protein has taken over a large role in substrate recognition
in this enzyme.
40
References
1.
Burgin, A.B. and N.R. Pace. 1990. Mapping the active site of ribonuclease P
RNA using a substrate containing a photoaffinity agent.Embo J 9(12), 4111-8
2.
Pace, N.R. and J.W. Brown. 1995. Evolutionary perspective on the structure and
function of ribonuclease P, a ribozyme. J Bacteriol 177(8), 1919-28
3.
Guerrier-Takada, C., K. Gardiner, T. Marsh, N. Pace and S. Altman. 1983. The
RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 35(3 Pt
2), 849-57
4.
Brown, J.W. 1999. The Ribonuclease P Database.Nucleic Acids Res 27(1), 314
5.
Stark, B.C., R. Kole, E.J. Bowman and S. Altman. 1978. Ribonuclease P: an
enzyme with an essential RNA component. Proc Natl Acad Sci U S A 75(8),
3717-21
6.
Gardiner, K. and N.R. Pace. 1980. RNase P of Bacillus subtilis has a RNA
component. J Biol Chem 255(16), 7507-9
7.
Vioque, A., J. Arnez and S. Altman. 1988. Protein-RNA interactions in the RNase
P holoenzyme from Escherichia coli. J Mol Biol 202(4), 835-48
8.
Loria, A. and T. Pan. 1997. Recognition of the T stem-loop of a pre-tRNA
substrate by the ribozyme from Bacillus subtilis ribonuclease P. Biochemistry
36(21), 6317-25
9.
Niranjanakumari, S., T. Stams, S.M. Crary, D.W. Christianson and C.A. Fierke.
1998. Protein component of the ribozyme ribonuclease P alters substrate
recognition by directly contacting precursor tRNA. Proc Natl Acad Sci U S A
95(26), 15212-7
10.
Stams, T., S. Niranjanakumari, C.A. Fierke and D.W. Christianson. 1998.
Ribonuclease P protein structure: evolutionary origins in the translational
apparatus. Science 280(5364), 752-5
11.
Green, C.J., R. Rivera-Leon and B.S. Vold. 1996. The catalytic core of RNase P.
Nucleic Acids Res 24(8), 1497-503
12.
Reich, C., G.J. Olsen, B. Pace and N.R. Pace. 1988. Role of the protein moiety of
ribonuclease P, a ribonucleoprotein enzyme. Science 239(4836), 178-81
13.
Chen, J.L. and N.R. Pace. 1997. Identification of the universally conserved core
of ribonuclease P RNA [letter]. RNA 3(6), 557-60
41
14.
Waugh, D.S., C.J. Green and N.R. Pace. 1989. The design and catalytic properties
of a simplified ribonuclease P RNA. Science 244(4912), 1569-71
15.
Christian, E.L., N.M. Kaye and M.E. Harris. 2000. Helix P4 is a divalent metal
ion binding site in the conserved core of the ribonuclease P ribozyme [In Process
Citation].Rna 6(4), 511-9
16.
Kleineidam, R.G., C. Pitulle, B. Sproat and G. Krupp. 1993. Efficient cleavage of
pre-tRNAs by E. coli RNAse P RNA requires the 2'- hydroxyl of the ribose at the
cleavage site. Nucleic Acids Res 21(5), 1097-101
17.
Kufel, J. and L.A. Kirsebom. 1996. Residues in Escherichia coli RNase P RNA
important for cleavage site selection and divalent metal ion binding. J Mol Biol
263(5), 685-98
18.
Warnecke, J.M., J.P. Furste, W.D. Hardt, V.A. Erdmann and R.K. Hartmann.
1996. Ribonuclease P (RNase P) RNA is converted to a Cd(2+)-ribozyme by a
single Rp-phosphorothioate modification in the precursor tRNA at the RNase P
cleavage site. Proc Natl Acad Sci U S A 93(17), 8924-8
19.
Oh, B.K. and N.R. Pace. 1994. Interaction of the 3'-end of tRNA with
ribonuclease P RNA. Nucleic Acids Res 22(20), 4087-94
20.
Nolan, J.M., D.H. Burke and N.R. Pace. 1993. Circularly permuted tRNAs as
specific photoaffinity probes of ribonuclease P RNA structure. Science
261(5122), 762-5
21.
Chamberlain, J.R., Y. Lee, W.S. Lane and D.R. Engelke. 1998. Purification and
characterization of the nuclear RNase P holoenzyme complex reveals extensive
subunit overlap with RNase MRP. Genes Dev 12(11), 1678-90
22.
Forster, A.C. and S. Altman. 1990. Similar cage-shaped structures for the RNA
components of all ribonuclease P and ribonuclease MRP enzymes. Cell 62(3),
407-9
23.
Stolc, V. and S. Altman. 1997. Rpp1, an essential protein subunit of nuclear
RNase P required for processing of precursor tRNA and 35S precursor rRNA in
Saccharomyces cerevisiae [corrected and republished in Genes Dev 1997 Nov
1;11(21):2926- 37]. Genes Dev 11(18), 2414-25
24.
Frank, D.N. and N.R. Pace. 1998. Ribonuclease P: unity and diversity in a tRNA
processing ribozyme. Annu Rev Biochem 67, 153-80
25.
Pannucci, J.A., E.S. Haas, T.A. Hall, J.K. Harris and J.W. Brown. 1999. RNase P
RNAs from some Archaea are catalytically active. Proc Natl Acad Sci U S A
96(14), 7803-8
42
26.
Nieuwlandt, D.T., E.S. Haas and C.J. Daniels. 1991. The RNA component of
RNase P from the archaebacterium Haloferax volcanii. J Biol Chem 266(9), 568995
27.
Darr, S.C., B. Pace and N.R. Pace. 1990. Characterization of ribonuclease P from
the archaebacterium Sulfolobus solfataricus. J Biol Chem 265(22), 12927-32
28.
LaGrandeur, T.E., S.C. Darr, E.S. Haas and N.R. Pace. 1993. Characterization of
the RNase P RNA of Sulfolobus acidocaldarius. J Bacteriol 175(16), 5043-8
29.
Haas, E.S., D.W. Armbruster, B.M. Vucson, C.J. Daniels and J.W. Brown. 1996.
Comparative analysis of ribonuclease P RNA structure in Archaea. Nucleic Acids
Res 24(7), 1252-9
30.
Darr, S.C., K. Zito, D. Smith and N.R. Pace. 1992. Contributions of
phylogenetically variable structural elements to the function of the ribozyme
ribonuclease P. Biochemistry 31(2), 328-33
31.
Siegel, R.W., A.B. Banta, E.S. Haas, J.W. Brown and N.R. Pace. 1996.
Mycoplasma fermentans simplifies our view of the catalytic core of ribonuclease
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32.
Siew, D., N.H. Zahler, A.G. Cassano, S.A. Strobel and M.E. Harris. 1999.
Identification of adenosine functional groups involved in substrate binding by the
ribonuclease P ribozyme. Biochemistry 38(6), 1873-83
33.
Zimmerly, S., D. Drainas, L.A. Sylvers and D. Soll. 1993. Identification of a 100kDa protein associated with nuclear ribonuclease P activity in
Schizosaccharomyces pombe. Eur J Biochem 217(2), 501-7
34.
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thermophila. J Biol Chem 271(28), 16559-66
43
Appendix 1 – TP/PT Construct
When I came into the lab as an undergraduate I worked on constructing and testing a
unimolecular enzyme:substrate RNA with the RNase P RNA from Methanobacterium
formicicum and pre-tRNAAsp from Bacillus subtilis. This construct was later used in the
paper Pannucci et al. “RNase P RNAs from some archaea are catalytically active.” The
original purpose of these complexes was to test the ability of RNase P RNA from the
Archaea to cleave without having to bind substrate. This was before we knew that some
of the archaeal RNase P RNAs (including that of Methanobacterium formicicum) were
active and could carry out the cleavage reaction under very high salt conditions. Using
PCR to amplify the RNase P RNA gene followed by ligation of the two ends allowed an
internal PCR reaction circularly permuting the gene such that the “active site” was at both
ends. This reordered RNase P RNA could then be placed in the pDW128 plasmid next to
the pre-tRNA. If the RNase P RNA (central loop) gene was placed at the 5′end of the
pre-tRNA it was referred to as the PT construct while the TP construct had the RNase P
RNA (attached L15)gene at the 3′ end of the pre-tRNA. Both constructs could have their
pre-tRNA cleaved off by E.coli RNase P RNA. However, the TP RNA could cleave
itself, but the PT RNA is not catalytically active. The conditions for this reaction were
almost identical to the RNase P RNA reaction requiring 300mM MgCl2 and 3M
NH4OAc. The reaction rate remained the same across increasing concentrations,
indicating the reaction is carried out in cis. The TP construct is now being used by D.
Williams in a SELEX experiment to identify mutants that might require lower salt
concentration.
44
G AA CA
U
A
U
G
GA AG A U
C
G
U
A GUC U C
G
GG
C
A A
U
U
U
C
C
G
U
C
A
U
ACGA
G
AA
C
UC
G
A G
AG
A
G
C
G
C
C
G AA
G
G
A
U
G
G
G
A
U
A
C
A
U
GA
C
A
C
GC
A
G
A
G
AG G U
GU
AACC
C
C
GU
G
C
C
G U AA
A
AA C
CG
A
U
A
A
U
G GAU G C A A GGGU
A A GU
CUGCC
C
G
C U
C
C
C CU A C
A G
C
U
C
A
G
A
C
G
G
U
A
G U
C
G
G
G
A
A
A
A
A
C
C
GU AU
C
U
GACAU
A
A C
A
U
A
G
C
G
3´
CU
U
G
UC
C
A
A
A
C U A G G A C C G C G U A A A A C C C A U G G C U C G G A G C U U A A G G G5
G
A
C G
A
A
G
A U
U
G
G C
G
U U
A
G C
A
A U GUCUGUAG AAACUG
C G
A
A
C GU
G
U
U U UAGAUAUC UUUGGC
A UC C C G U
A
AG U
G
C
"
C UG
GUUC
G
U
C
A
"
C U GG G C G
G
U
A
U
UAAG
G
UC
G
C
AUU
G
G AU C
G GA G C
C
G
G
C
G C
A
G
ACCGGGCA GCCGAAGG
C A C C AA C A
A U
G
C
C G
A
C
UGGCCCGUA CGGCUUC
Asp
G C
G
C
A
C
C
C A
A G AU
UU GG G U G G
U
A
CU G
Methanobacterium formicicum
cpTP RNA
P9
L15
Cleavage Site
P8
P6
P3
P4
Bacillus subtilis pre-tRNA
P1
GAA CA
U
A
U
G
GA AG A U
C
G
A GU C U C
GU
G
G
C
U
A A
U
C
U
G
U
CC
A
ACGA
CU
G
AA
C
U
G
A G
AG
A
C
GG
C
G CA A
G
G
A
U
G
G
G
A
U
A
C
A
U
G AA
C
C
G
A
G
A
CA
G
GU
GG UA A C C
C
C
GU
GA
CA
C
GU
A
AAC
CG
U
A
A
A
U
G GAUG C A A GGGU
A A GU
UGCC
C
C
G
C U
CC
C
A G
CU A C
CUCA
GACGG
U
A
G U
C
G
G
G
A
A
A
A
A
C
C
U
U
G
GU A
G
C
GACAU
A C
A
U
A
C
U G G U A C C C A A A A U G C G C C A 3´
UC
C
G C
A
A
U A
A
AGA C U U AA GGG
C G
5´
G
U
C G
G
G
G C
U U
A
A
UG C
A U GUCUGUAG AAACUG
G
A
A
A
U G C C CU
G
A
U A
"
G
U
CUUG
U
U U UAGAUAUC UUUGGC
GC GG G
"
G
C
C
G
U
C U
U
C
U
G
A
A
C
G
U
GU A
A
G
U
U
A
C G AGG
G
C G
C
G AU C
U A
C
G
G
C
G C
G
C A C C AA C A
AC CGG G CA A G CC GAA GG
C G
G
C
C
C
A
C
UGGCCCGUACGGCUUC
U
A
A
G
C A
C
G
C
U
A G AU
UU GGG U G G
Methanobacterium formicicum
cpPT RNA
P9
L15
P8
P3
P6
Cleavage Site
P4
P1
Asp
Bacillus subtilis pre-tRNA
Figure 18. Secondary structures for both the TP and PT constructs.
45
0.06
100
A.
nM remaining
75
Activity (%)
B.
0.05
50
0.04
0.03
0.02
25
1mM spermidine
0.01
no spermidine
0
0
1
3
4
5
0
0
NH4+ (M)
C.
2.5
2
100
D.
100
nM cleaved
200
300
400
Time (min)
90
Activity (%)
75
50
1
25
0.5
Activity (percent)
1.5
80
Activity (%)
2
nM cleaved
100
70
60
50
40
30
20
10
0
0
0
1
2
3
nM added
4
5
0
6
6.5
7
7.5
8
8.5
pH
Figure 19. Results from TP assays; A. TP activity with and without spermidine as
function of NH4+, B. TP activity as a function of time, C. Rate of TP activity as a function
concentration. D. TP activity across a pH gradient. All assays were done in 300mM
MgCl2 and 3M NH4OAc for 4hr. at 60°C unless otherwise stated.
46