RNase P in Archaea
James W. Brown
NCSU Microbiology
Looking to a warm place for P
Brown lab
Department of Microbiology
North Carolina State University
RNase P is the pre-tRNA 5´endonuclease
RNase P assay
- +
NCSU Microbiology
Brown lab
RNase P is present in all cells, and in mitochondria and plastids
Why study RNase P?
•
RNase P is apparently a leftover of the ‘RNA World’,
and so can teach us about the emergence of life and
the origin of Bacteria, Archaea and Eukaryotes
•
RNase P is a ribozyme (RNA enzyme), and so can
teach us about RNA structure and function
•
RNase P is highly conserved in Bacteria, and very
different in eukaryotes, and like the ribosome is a
large essential RNP, and so has great potential as a
target for novel antimicrobials
•
RNases I, II, III, A, B, E, G, H, P1, P2, PH, S, T1, T2,
U1, U2, V1, and Z were already taken
NCSU Microbiology
Brown lab
RNase P in Bacteria
product
mature tRNA
~76nt/24kDa
(green)
1 large RNA
rnpB
377nt/140kDa
(blue & purple)
1 small protein
rnpA
119aa/14kDa
(red)
NCSU Microbiology
Brown lab
The RNA by itself is catalytically proficient in vitro
RNase P in Eukarya1898(yeast nucleus)
One large RNA
Rpr1 - 120kDa
This RNA is only generally
similar to the bacterial RNA
Nine assorted proteins
Pop1p - 100kD
Pop3p - 23kD
Pop4p - 33kD
Pop5p - 20kD
Pop6p - 18kD
Pop7p - 16kD
Pop8p - 15kD
Rpp1p - 32kD
Rpr2p - 16kD
None of these are at all
similar to the bacterial protein
NCSU Microbiology
Brown lab
The RNA is absolutely dependent on the proteins for activity
The three evolutionary Domains
cyanobacteria
Gram-positive
proteobacteria
spirochaetes
planctomycetes
Thermotoga
Aquifex
Crenarchaea
Euryarchaea
diplomonads
flagellates
amoebas
animals
fungi
plants
Bacteria
Archaea
Eukarya
NCSU Microbiology
Brown lab
Archaea are an ideal “missing link” for understanding the evolutionary
differences between Bacteria and eukaryotes
RNase P in Archaea - a Preview
A bacterial-like RNA...
rnpB - 96kDa (293nt)
Ribonuclease P RNA
Methanobacterium thermoautotrophicum ΔH
11
1618
688
687
A catalytically-active “type A” RNA in most cases,
only distantly related to the eukaryotic RNA
Sequence : U42986, Pannucci 1999 PNAS 96:7803
Structure : Harris, et al., RNA (in press)
Image created 10/5/00 by JWBrown
G
U
A
G
UG
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A A
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A
A
G CU
U C A CC
A
CGA
C
G
G
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A
U
CU
A CA
C
U
G
A
C
G AC
C
A
GUC A
UU
AC
A G U
GA
A
C
AG
A
G
P12
G
A
G CC A
A
G
C
U
G
G
A
A
G G
A
G
G C A
C C U CU
A
G
P9 C
A
GG
P10/11
A
C U GA U U
GU
G
CG
C
AC
C A P7
CG
P15 A A A U G P16 G G U P17
P5
U
GG AUG C A A GG AC
CUGCC
GA U
A
G GU
A
GC
U
P8 C U
C
C
G
G
C
U
U
G
G
A
U
G
G
A
U
C
U
A
A
C
G
G
C
G
A
GU
A
AG
A
G
C
UGG
A
A CAU
C
A
C
C
GUG
A
C
C
C
C
A
U
C
G
A
A
A
U
A
G
G
P6
G
A
P3
A
A
A
A GGGGCUG
U
U
C C C C U G G C G C G P4
U
A G C
A U
P2 C G
P1
G C
5´ A G C C G A A G G C G A A A C
A
CGGCU UC
G
3´ A
C A
A
A
UUGGGUGG
but 4 eukaryotic-like proteins.
RNase P in Archaea is an evolutionary chimera
MTH687p - 15kDa = Pop5p
MTH688p - 28kDa = Rpp1p
MTH11p
- 11kDa = Pop4p (C-terminal half)
MTH1618p - 17kDa = Rpr2p
Not at all like the single bacterial protein
NCSU Microbiology
Brown lab
The Archaea
Haloferax
Euryarchaea
Extreme halophiles
Methanosarcina
Thermoplasma
Methanothermobacter
Archaeoglobus
Methanocaldococcus
Pyrococcus
Crenarchaea
Thermoproteus
Pyrodictium
Acidianus
Methanogens
Sulfur-metabolizing
thermophiles
Sulfolobus
NCSU Microbiology
Brown lab
Phylogenetic tree of the Archaea based on ssu-rRNA sequence analysis
NCSU Microbiology
Brown lab
NCSU Microbiology
Brown lab
NCSU Microbiology
Brown lab
NCSU Microbiology
Brown lab
RNase P in Archaea : Early Information
Haloferax volcanii
Sulfolobus acidocaldarius
1.61 g/cm3 in Cs2SO4
1.27 g/cm3 in Cs2SO4
rapid pelleting in glycerol
large exclusion radius (400kDa)
nuclease sensitive
nuclease resistant
copurifying 435nt RNA
copurifying 315nt RNA
(but also reported to be inactivated)
(suggests protein alone)
(degraded by nuclease treatment)
only vaguely similar to other P RNAs
(not degraded by nuclease treatment)
only vaguely similar to other P RNAs
RNA not catalytically active
RNA not catalytically active
Early reports of catalytic activity by the RNA alone
and reconstitution with bacterial protein
could not be reproduced and were discounted
The RNase P RNAs of Methansarcina barkeri and Methanocaldococcus jannaschii are also not
catalytically active, leading to the conclusion that archaeal RNase P RNAs, like those of eukaryotes,
absolutely depend on protein for function.
NCSU Microbiology
Brown lab
Our first task, then was to identify the differences in bacteria and archaeal
RNase P RNAs that might be responsible for this difference in activity.
Archaeal RNase P RNA 2° structures
CA
U
C
G G
G U
C G
C
A
C
C
A CC
G
G
C
GA
G
G
C
Sequence : M61003, Neuwlandt, et al, 1991 J. Biol. Chem. 266:5689
G
G
AA C
C
C
Structure : Harris, et al., RNA (in press)
GAA
G G G
G
C
C G CC
U GC
G
G U CG
G
C
G A
Image created 10/5/00 by JWBrown
C CG
C
U CG G
CA G C
U
G GC
A
C G
A
C
C
G GC C
A UG AU
C
G
A
C
G
C
A
A
G
G
G
C
C
G
C
A
GA
G
U
UC
G
UG C A
C
G
A
G CC
GA
A
G
GC
AC
C
U G
A
C
GA
C G
C A
A
U
U
GG A
G
A G
A
A
C G
U
C
C
C
G
G
A
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CU
A
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A
G
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CU
G
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G AC
C
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GG
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A
U
GGG
CU G
C
GG
AA
U
CU
C
G
A
GA
C
UC
G
A
G
G
C
U
C G G UG C A A G U C CG CG C C
G
C AG
U
U
C
C
GG
C
C G
CCAC
C A G G CG CG G
G
G
C G
C AA
G
A
C
G
G
G CC
G G
A
U C
A
A
C
C
G
C
C
A G
A
C
G
C
U
C
C
G
U C G
G
C
C
C
G C
C
U
G
A
C G
G
C G
A
G C
G
C G
G
C G
A
G
C
C
U
U
A
G
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U G
G C
G
C
G
C
G
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C G
A
A
C G
A
A CG G G C A UG
U
G
G
CU
U
A G C C CG UG C U
C
C U G
G C
A U
C G
5´
C G
GG CA G AG AG AG C G A CG A A
C
3´
A
U U C G C CG A C U C U C U C
G
C
A
A
A
UU CGGG GG
Ribonuclease P RNA
Haloferax volcanii DS-2
Ribonuclease P RNA
Sulfolobus acidocaldarius(solfataricus) P1
Sequence : L13597, LaGrandeur, et al, 1993 J. Bact 175:5043
Structure : Harris, eet al., RNA (in press)
Image created 10/5/00 by JWBrown
A
UG
A AA
A
U
G
A
G U
UU A
UA
UG
A
AA
A
U
UA
U
U
A
A
A
C
UA
GU
U
G
U
CAGA
U
C
CU
G
AU
A
CG U A A
AG
U
CG
A
CG
G
A
C
GA
A
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U
C
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A
G
C
G
A A
G
C
A
U
C
G
A
A
C
G G G C AA
U
C
A
U C
A
GU
G
C
G
A
CU
G
U
CU G
UU
C
GG
C
GA
UC
C
G AU
C
CA
G G AG
GG AG C A A G U A AGG AGGG G
G
U
C
A U UG
GU
G
C
A
A
CCUC
CA
UCCUCCC A
A
U
G
G
A
C
A
A
G
U
A
C
G
C G
C CG
U
U
G A
A G
GA U
U
C
A
U
C
U
C
U
A
C
G
A
A
U
A
A
G
G
G
A
A
A U C C UG
A
U
U
A A G G G C UU
C
A G
G C
G C
G C
G C
5´
UA A
U AGGGG AG CCUA A C A
A U A
C
3´
U C C C C U CG G A U UG
A
UAU
NCSU Microbiology
U
A
U
A
A
U UGGG U CG A
Determined to base-pair resolution by comparative sequence analysis
U
A
G
G
UA
Brown lab
Archaeal vs bacterial RNase P RNAs
Ribonuclease P RNA
Methanobacterium thermoautotrophicum ΔH
Sequence : U42986, Pannucci 1999 PNAS 96:7803
Structure : Harris, et al., RNA (in press)
Image created 10/5/00 by JWBrown
G
U
A
G
UG
AU
A A
U
G
A
A
G CU
U C A CC
A
CGA
C
G
GG
U
A
U
A CA
CC
U
G
A
C
G
U
AC
C
A
GUC A
U
AC
A G U
GA
A
C
AG
A
G
P12
G
A
G CC A
A
G
C
U
G
G
A
A
G G
A
G
G C A
C C U CU
G A
P9
C
A
GG
P10/11
A
C U GA U U
GU
G
CG
C
AC
C A P7
CG
P15 A A A U G P16 G G U P17
P5
U
G
GG AUG C A A GG AC
CUGCC
GA U
A
G U
A
GC
U
P8 C U
C
C
G
G
C
U
U
G
G
A
U
G
G
A
U
CU AC
A
G
G
C
G
A
G
A
AG
A
G
U
UGG
A
A CAU C
C
A
C
C
GUG
A
C
C
C
C
A
U
C
G
A
A
A
U
A
G
G
P6
G
A
P3
A
A
A
A GGGGCUG
U
U
C C C C U G G C G C G P4
U
A G C
A U
P2 C G
P1
G C
5´ A G C C G A A G G C G A A A C
A
CGGCU UC
G
3´ A
C A
A
UUGGGUGGA
UU
C
G
G
G
C
G
C
C
C
C
G
G
G
C
C
C
G
G
G
U
U
AG
AG C
G
A
C
U
A
G
Ribonuclease P RNA
G
G
G
Ralstonia (Alcaligenes) eutrophus DSM 531
A
G
U
A
G
Sequence : M59353, Brown, et al., 1991 J. Bacteriol. 173:3855
A
C
Structure : Harris, et al., RNA (in press)
A
A
C
A
C G CGC
Image created 10/3/00 by JWBrown
G G
U
A G
A
U G
C
A
C A
A CG UG C A A
G
U
C
G
C
GGCAC
AA UA
C
CA
A
G
G AU
CC
U
U
G CAGG C G
GG AG C A A UCCC
G
A
U
U
GG
A
C
U
AG
G CCUC
CG U C UG A
AGGG
C G
C GG
A
G
A
G
G G
A
A
G
C
C
U
UGG
A
C
C
G
C
G C
G
C
C
G A
C
U
G
U
A A
G
GG
C
A G C CG G C UG G
C
U
U
G
A
C
G
G
C
C
G
A
C
A
C
A
AU
A
G
A
A
G
G
G
G CA
G
C
A
A
A
G
C
A
G
G
A G G UG C A G G G G
A
G
U
C CCAC
U
C
U
G U C CG U C G
G
G
C G
U
G
G
C G
G
C
A U
C
AC G
A U
G
C
C G
5´
G U CG G G
C
A A AG CAGG CC A G C A
G
3´
NCSU Microbiology
C
A
CA
U U C G U U U CG U C CG G U
G
CA
A
U U CGG C C U A
The type A structure is common to most Archaea and Bacteria
Brown lab
Some archaeal RNase P RNAs are active!
NR Eco Mse Sac Pfu Tce Mja Mth Mma Mva Mfo ERH MtM ERE MtD Mba Hvo Ngr
pre-tRNA
mature tRNA
5´leader
NCSU Microbiology
Brown lab
Assays contain 4M NH4OAc, 300mM MgCl2, a 200:1 ratio of enzyme to substrate,
incubated for 3.5 hr at 45°C. In vivo or in vitro RNA.
Properties on archaaeal RNase P enzymes
Methanothermobacter thermoautotrophicus
Property
RNA-alone
Holoenzyme
Km
>50,000nM
34.5±3.5nM
kcat
>2.2/min
52.6±4.3/min
density
1.65
1.42
Topt
~50°C
≥80°C
[NH4OAc]opt
3.0M
0.8M
[MgCl2]opt
300mM
5mM
exclusion volume
(nd)
450kDa
The archaeal RNA mimics the properties, including general lead cleavage
sensitivity, of bacterial RNAs lacking peripheral stabilizing elements, but the fact
that the RNA contains any activity implies that they have all of the sequences
and structures required for substrate recognition and catalysis.
NCSU Microbiology
Brown lab
Chimeric holoenzymes are active
Functional reconstitution is reflected in the enhancement of activity in the presence of
protein in assays performed at low ionic strength
no P RNA
rotein
Bsu P p
E.coli P RNA
BSA
rotein
Bsu P p
H.volcanii P RNA
BSA
rotein
Bsu P p
M.formicicum P RNA
BSA
rotein
Bsu P p
M.thermo. P RNA
BSA
rotein
Bsu P p
pre-tRNA
mature tRNA
5´leader
NCSU Microbiology
Brown lab
Archaeal RNase P RNAs can bind the Bacillus subtilis RNase P protein, and the
chimeric holoenzymes are functional!
Archaea ought to have a bacterial-like protein
LOCUS
B.aphidocola
114 bp
DEFINITION B.aphidocola
114 b, 114 bases, 58069AB0 checksum.
ORIGIN
1 MLNYFFKKKS KLLKSTNFQY VFSNPCNKNT FHINILGRSN LLGHPRLGLS
51 ISRKNIKHAY RRNKIKRLIR ETFRLLQHRL ISMDFVVIAK KNIVYLNNKK
101 IVNILEYIWS NYQR
//
LOCUS
B.burgdorferi
119 bp
DEFINITION B.burgdorferi , 119 bases, E73BF549 checksum.
ORIGIN
1 MRKRNISLKS KIEIQKIFKE GKLIRFSNLN LKMFYKSNHL VYSRILVTFS
51 KGFRGSVKRN RIRRLFKEAF RKRLELLEGI ALDIIFVVSY GKLTLTYFSI
101 ESLMKGLVLR CERGIGESK
//
LOCUS
B.halodurans
118 bp
DEFINITION B.halodurans
, 118 bases, FC638DB3 checksum.
ORIGIN
1 MKKEHRIKRS DEFSRVFNEG FSVANRQFVI YVLPKEGQDF FRVLSVSKKI
51 GNAVTRNRVK RLIRTFFQEH EQAISGERDY VIIARKPAAD MTYEQVKGSL
101 WHVCKKAKII QPKVRAHK
//
LOCUS
B.subtilis
119 bp
DEFINITION B.subtilis
119 bp, 119 bases, 724961C checksum.
ORIGIN
1 MSHLKKRNRL KKNEDFQKVF KHGTSVANRQ FVLYTLDQPE NDELRVGLSV
51 SKKIGNAVMR NRIKRLIRQA FLEEKERLKE KDYIIIARKP ASQLTYEETK
101 KSLQHLFRKS SLYKKSSSK
//
RBS
P>
P >P >
dnaA
rpmH
RBS
yidD
rnpA
TT
NCSU Microbiology
Brown lab
...but, they don’t!
RNase P in Eukarya1898(yeast nucleus)
One large RNA
Rpr1 - 120kDa
This RNA is only generally
similar to the bacterial RNA
Nine assorted proteins
Pop1p - 100kD
Pop3p - 23kD
Pop4p - 33kD
Pop5p - 20kD
Pop6p - 18kD
Pop7p - 16kD
Pop8p - 15kD
Rpp1p - 32kD
Rpr2p - 16kD
None of these are at all
similar to the bacterial protein
NCSU Microbiology
Brown lab
Proteins identified in Dave Engelke’s lab
RNase P in Eukarya1898(yeast nucleus)
One large RNA
Rpr1 - 120kDa
This RNA is only generally
similar to the bacterial RNA
Nine assorted proteins
Pop1p - 100kD
Pop3p - 23kD
Pop4p - 33kD
Pop5p - 20kD ~Mth687p
Pop6p - 18kD
Pop7p - 16kD
Pop8p - 15kD
Rpp1p - 32kD
Rpr2p - 16kD
None of these are at all
similar to the bacterial protein
NCSU Microbiology
Brown lab
Archaea encode a protein with vague similarity to yeast nuclear P protein Pop5p!
RNase P in Eukarya1898(yeast nucleus)
One large RNA
Rpr1 - 120kDa
This RNA is only generally
similar to the bacterial RNA
Nine assorted proteins
Pop1p - 100kD
Pop3p - 23kD
Pop4p - 33kD ~Mth11p
Pop5p - 20kD ~Mth687p
Pop6p - 18kD
Pop7p - 16kD
Pop8p - 15kD
Rpp1p - 32kD ~Mth688p
Rpr2p - 16kD ~Mth1618p
None of these are at all
similar to the bacterial protein
NCSU Microbiology
Brown lab
Archaea encode proteins with vague similarity to 4 yeast nuclear P proteins!
All 4 proteins copurify with P activity
Westerns using antisera specific to each protein reveal that all 4 proteins
copurify exactly with RNase P activity in fractions from glycerol gradients of
partially-purified RNase P.
NCSU Microbiology
Brown lab
Activity can be reconstituted from the archaeal
RNA and these 4 proteins
Kouzuma, et al., (Makoto Kimura lab) 2003 BBRC 306:666-673
Mth687p
Mth1618p
Mth11p
Mth688p
NCSU Microbiology
Brown lab
This is the Pyroccoccus horikoshi system, but it also works in
Methanothermobacter thermoautotrophicus.
Archaeal vs yeast P subunit interactions
RNA
Pop1
RNA
Pop3
?
11
1618
688
687
Methanothermobacter
Pop7
Pop4
Rpr2
Pop6
Rpp1
Pop5
Pop8
Saccharomyces
NCSU Microbiology
Brown lab
The interactions between homologous components are conserved
The archaeal proteins are not similar in
structure to the bacterial P protein
Mth11p/Pop4p
(Archaeoglobus fulgidus)
an oligonucleotide-binding β-barrel
Mth688p/Rpp1p
(Pyrococcus horikoshii)
a TIM metallohydrolase α/β-barrel
bacterial rnpA
(Thermotoga maritima)
an EF-G α/β sandwich
Mth1618p/Rpr2p
(Pyrococcus horikoshi)
α-helix/Zn-ribbon
Mth687p/Pop5p
(Pyrococcus furiosus)
an RNR α/β-sandwich
This argues against any underlying homology between the bacterial and any of
the archaeal proteins
NCSU Microbiology
Brown lab
Possible evolutionary scenarios : RNA
Bacteria
A
?
RNA
World
LCA
Archaea
A
E
Eukarya
NCSU Microbiology
Brown lab
This scenario is uncontroversial
Possible evolutionary scenarios : Protein
Bacteria
rnpA
?
RNA
World
LCA
Archaea
Pop
Eukarya
NCSU Microbiology
Brown lab
In this scenario, the single small bacterial protein represents evolutionary
streamlining from a more complex archaeal-like primitive state
Possible evolutionary scenarios : Protein
Bacteria
rnpA
?
RNA
World
LCA
Archaea
Pop
Eukarya
NCSU Microbiology
Brown lab
In this scenario, the LCA is closer to the RNA World, with an RNA-only RNase P,
and the 2 emerging branches recruited proteins independently
RNase P in Archaea - a Review
A bacterial-like RNA...
rnpB - 96kDa (293nt)
Ribonuclease P RNA
Methanobacterium thermoautotrophicum ΔH
11
1618
688
687
A catalytically-active “type A” RNA in most cases,
only distantly related to the eukaryotic RNA
Sequence : U42986, Pannucci 1999 PNAS 96:7803
Structure : Harris, et al., RNA (in press)
Image created 10/5/00 by JWBrown
G
U
A
G
UG
AU
A A
U
G
A
A
G CU
U C A CC
A
CGA
C
G
G
G
A
U
CU
A CA
C
U
G
A
C
G AC
C
A
GUC A
UU
AC
A G U
GA
A
C
AG
A
G
P12
G
A
G CC A
A
G
C
U
G
G
A
A
G G
A
G
G C A
C C U CU
A
G
P9 C
A
GG
P10/11
A
C U GA U U
GU
G
CG
C
AC
C A P7
CG
P15 A A A U G P16 G G U P17
P5
U
GG AUG C A A GG AC
CUGCC
GA U
A
G GU
A
GC
U
P8 C U
C
C
G
G
C
U
U
G
G
A
U
G
G
A
U
C
U
A
A
C
G
G
C
G
A
GU
A
AG
A
G
C
UGG
A
A CAU
C
A
C
C
GUG
A
C
C
C
C
A
U
C
G
A
A
A
U
A
G
G
P6
G
A
P3
A
A
A
A GGGGCUG
U
U
C C C C U G G C G C G P4
U
A G C
A U
P2 C G
P1
G C
5´ A G C C G A A G G C G A A A C
A
CGGCU UC
G
3´ A
C A
A
A
UUGGGUGG
but 4 eukaryotic-like proteins.
RNase P in Archaea is an evolutionary chimera
MTH687p - 15kDa = Pop5p
MTH688p - 28kDa = Rpp1p
MTH11p
- 11kDa = Pop4p (C-terminal half)
MTH1618p - 17kDa = Rpr2p
Not at all like the single bacterial protein
NCSU Microbiology
Brown lab
Type M RNAs lack essential structure
Ribonuclease P RNA
Methanococcus jannaschii JAL-1
Ribonuclease P RNA
Methanobacterium thermoautotrophicum ΔH
Sequence : U42986, Pannucci 1999 PNAS 96:7803
Structure : Harris, et al., RNA (in press)
Image created 10/5/00 by JWBrown
G
U
A
G
UG
AU
A A
U
G
A
A
G CU
U C A CC
A
C
C
G
A GG
UG
C
A
U
CC
UA A
G
A AC
G AC
U
C
C
GUC A
U
A
A G U
GA
A
C
AG
A
G
P12
G
A
G CC A
A
G
C
U
G
G
A
A
G G
A
G
G C A
C C U CU
A
G
P9 C
A
GG
P10/11
A
C U GA U U
GU
G
CG
C
AC
C A P7
CG
P15 A A A U G P16 G G U P17
P5
U
GG AUG C A A GG AC
CUGCC
GA U
A
G GU
A
GC
U
P8 C U
C
C
G
G
C
U
U
G
G
A
U
G
G
A
U
CU AC
A
G
G
C
G
A
GU
A
AG
A
G
C
UGG
A
A CAU
C
A
C
C
GUG
A
C
C
C
C
A
U
C
G
A
A
A
U
A
G
G
P6
G
A
P3
A
A
A
A GGGGCUG
U
U
C C C C U G G C G C G P4
U
A G C
A U
P2 C G
P1
G C
5´ A G C C G A A G G C G A A A C
A
CGGCU UC
G
3´ A
C A
A
UUGGGUGGA
P8
T-loop binding
A CG
G
A
U
U
A
G
A
G
UA
A
Image created 10/5/00 by JWBrown
GU
G
G
A
GU
A
A
CA GA
UG
G
U
G
A
G
A
U
C
C
C
C
U
G
C
G
A
A C A GG G
CG
C
C A
A
C A
A
G
A
G
G
U
A
G
C
A
G
A
A
A
C
C
GG
G G
C
U
A
A
U
G
C
A
U
G
GAA
C
G
GG
A
C
G
G
G
G U
C
C
CC
CU
CC
GC
G
CC
U C G
G G G G UG C A A G C CG G
AC
GG
U
GU
CCCAC
CG G C UU
A
C
C
G
U
C
U UA
C
G
U
C
U
C
U
A
U
G
G
A
C
A
C
G
G
G
A
A
A AG AGGGG
A
U
U
U UCUCCCC U
U
U C G
A U
G C
U A
G C
5´ A
GGGGG CU G CG A A A U U A
C
A
C C C C CG A
3´ A
G
U
A U CGGG CGG AA
Sequence : L77117, Bult, et al., 1996 Science 273:1058
Structure : Harris, et al., RNA (in press)
L15
3´NCCA binding
P8?
L15?
NCSU Microbiology
Brown lab
Our hypothesis is that in type M enzymes, protein sequences directly replace
the structure and functions lost in the RNA.
Pyrobaculum RNase P lacks essential structure
Ribonuclease P RNA
Methanobacterium thermoautotrophicum ΔH
All 4 RNase P protein-encoding
genes are also absent
Sequence : U42986, Pannucci 1999 PNAS 96:7803
Structure : Harris, et al., RNA (in press)
Image created 10/5/00 by JWBrown
G
U
A
G
UG
AU
A A
U
G
A
A
G CU
U C A CC
A
C
C
G
A GG
UG
C
A
U
CC
UA A
G
A AC
G AC
U
C
C
GUC A
U
A
A G U
GA
A
C
AG
A
G
P12
G
A
G CC A
A
G
C
U
G
G
A
A
G G
A
G
G C A
C C U CU
A
G
P9 C
A
GG
P10/11
A
C U GA U U
GU
G
CG
C
AC
C A P7
CG
P15 A A A U G P16 G G U P17
P5
U
GG AUG C A A GG AC
CUGCC
GA U
A
G GU
A
GC
U
P8 C U
C
C
G
G
C
U
U
G
G
A
U
G
G
A
U
CU AC
A
G
G
C
G
A
GU
A
AG
A
G
C
UGG
A
A CAU
C
A
C
C
GUG
A
C
C
C
C
A
U
C
G
A
A
A
U
A
G
G
P6
G
A
P3
A
A
A
A GGGGCUG
U
U
C C C C U G G C G C G P4
U
A G C
A U
P2 C G
P1
G C
5´ A G C C G A A G G C G A A A C
A
CGGCU UC
G
3´ A
C A
A
UUGGGUGGA
S-domain
substrate specificity
S-domain?
Putative RNase P RNA
Pyrobaculum aerophilum
AAA
U
Rough draft secondary structure
A
G
A
7/13/06 Jim Brown / Todd Lowe
A
G
C
A GG C
G C P10
G
A
G
A
G C CG
GC
CC
C G P7
G
P8
GG
G G C GG
GG
P5
P15 A A G C A P16 U G U
CU
C
G G GC C G
CU
GCGGC C CCC
A GC
CG
G
C
G
CCCGG A
CGCC
GG G
G
G
A
AU GG
C
A
G
C
C
G
G G
G
C
C
G
P17
G CG
G
U
C
G
P6
C
A
CR I U C
G
A CR IV
C
U
C
AA
A
G
G
P3
C
A
A
G CG G G C C C UU C U G
U
P4
A GC C C G G G GG G G C G A
G
A
A GG C
G C P2
G
P1
A
A A CR V
U A A C A G U U U A A A U G G GG C G C CG
C A
c cc g C G GC
G
A
ga a a a u g g c a u u a a c
G
A
C
AU G G G G G C G
NCSU Microbiology
Brown lab
But each tRNA gene is directly preceded by a promoter; so what is the substrate?
RNA Ontology and the RNase P Database
NCSU Microbiology
Brown lab
Acknowledgements
Chris Ellis
Tom Hall
Jim Pannucci
Dan Williams
Foster lab
Kimura lab
Andy Andrews
Kirk Harris
Jim Brown
Elizabeth Haas
Ginger Muse
John Gantt
Carl
Woese
Norm
Pace
Jeff Barnes
Bev Vucson
Sylvia
Darr
Hoffman lab
Ford
Dolittle
Tom LaGrandeur
Chuck
Daniels
Dan Nieuwlandt
NCSU Microbiology
Leo Thompson
$ NIH, NSF, Isis Pharmaceuticals
Brown lab
Fall 2006 Meeting
North Carolina Branch of the
American Society for Microbiology
The JC Ralston Arboretum
NC State University, Raleigh, NC
Thursday, October 12, 2006
Abstract deadline : September 28th
Registration deadline : Oct 5 (or at the door)
Registration fee + 2006 NC ASM membership
+
The featured speaker this year will be Dr. William Shafer from the Emory University School
of Medicine
For more info, visit our web site at:
NCSU Microbiology
Brown lab
http://www.asm.org/branch/brnc/index.html