Transcript Motion in RNA and DNA Polymerases

Motion in RNA and DNA
Polymerases
My Structural Biology Theology
Understanding the structural basis of a
biological process requires structures of the
assembly that carries out the process at each
step in the process.
How do polynucleotide
polymerases initiate synthesis at a
specific site and then synthesize
six to ten nucleotides while still
attached to the initiation site?
What is the nature of the initiation
to elongation transition?
Phage phi29 DNA polymerase:
protein primed initiation of DNA
synthesis
The structural basis of protein priming
OH
TTTCAT
Protein Priming
A
TTTCAT
Sliding Back Initiation
A
TTTCAT
Nucleotide Priming
AA
TTTCAT
Sequential Insertion of nts 3-5
AAAGT
TTTCAT
Sequential Insertion of nts 6-10
Dissociation of pol from TP
AAAGTANNNN
TTTCATNNNN
Elongation
How does the DNA polymerase
Extend the primer strand while
Still being bound to the priming
Terminal protein?
The Structure of f29 DNA polymerase
Exo
Thumb
TPR2
Fingers
Palm
TPR1
5
190
260
*
357
393 425
529
*
575
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Primer/template DNA and terminal protein have overlapping binding sites
Exonuclease
Thumb
TPR2
Template
Priming
domain
Primer
Palm
Intermediate
domain
TPR1
N-terminal
domain
Polymerase + primer/template + incoming nucleotide
Polymerase + terminal protein
Domain organization of terminal protein
Four-helix bundle domain
234
259
226
119
140
Hinge
Intermediate domain
N-terminal domain
Spiral translocation of DNA
+5
+2
template
primer
+8
incoming dNTP
a-phosphate
Addition of the first nucleotide
Four-helix
bundle domain
Hinge
Intermediate
domain
incoming dNTP
a-phosphate
Priming loop
N-terminal
domain
Addition of 2-4 nucleotides
+2
Four-helix
bundle domain
Hinge
Intermediate
domain
N-terminal
domain
Addition of 5-6 nucleotides
+5
Four-helix
bundle domain
Hinge
Intermediate
domain
N-terminal
domain
Addition of 7-8 nucleotides: hinge movement insufficient
Four-helix
bundle domain
Hinge
Intermediate
domain
N-terminal
domain
+8
Addition of 7-8 nucleotides: hinge movement insufficient
Four-helix
bundle domain
Hinge
Intermediate
domain
N-terminal
domain
+8
26 Å 35 Å
T7 Phage RNA Polymerase, a
DNA Dependent RNA
Polymerase
Template directed transcription
Upstream
Promoter
RNAP
Downstream
Transcription bubble formation
Promoter
RNAP
Initiation
Downstream
Upstream
RNAP
Promoter
5’RNA
Elongation
Downstream
Upstream
RNAP
Promoter
5’RNA
Initiation complex
NT 5’TAATACGACTCACTATA
Initiation complex 5’
T 3’ATTATGCTGAGTGATATCCCTC
+ GTP
+ T7 RNAP
NT 5’TAATACGACTCACTATA
T 3’ATTATGCTGAGTGATATCCCTC 5’
RNA
5’ GGG
3’
Designed substrate for elongation complex
ACACGTTACGT TGCGCACGGC
NT: 5’ GACAGGCTC
ACGCGTGCCG
T 3’ CTGTCCGAG
GTGTGCCGCTT
NT 5’ TAATACGACTCACTATA
CACACGGCGA
5’UUUUUGA
T 3’ ATTATGCTGAGTGATATCCCTC 5’
5’ GGG 3’
NT
T
Initiation to elongation phase structural transition in
T7 RNA polymerase
Cheetham and Steitz, 1999
Yin and Steitz, 2002
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Transition from initiation to elongation
- Abolishes the promoter recognition site
- Creates a DNA-RNA heteroduplex channel
Initiation
Elongation
Transition from initiation to elongation
- Creates an RNA exit tunnel
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Similar molecular strategies in other systems: prokaryotic
multisubunit RNA polymerases (T.th. and T.aq.)
Murakami and Darst, 2003.
Similar molecular strategies in other systems: eukaryotic
multi-subunit RNA polymerases (S.c. pol II)
Bushnell et al., 2003.
Similar molecular strategies in other systems: eukaryotic
multi-subunit RNA polymerases
Bushnell et al., 2003.
Different mechanisms, similar principles
•
•
•
The initiation factor is involved in recognition of the start site.
The polymerase synthesizes a bit of the new strand while the initiation
factor is still holding on to the initiation or recognition site.
The elongating duplex or transcript pushes the initiation factor out of
the polymerase active site.
System
Initiation factor
Recognition site
Phi29 DNA pol
Terminal protein
ori
T7 RNAP
N-terminal domain
promoter
Prokaryotic multisubunit RNAP
Sigma factor
promoter
Eukaryotic multisubunit RNAP
TFIIB
promoter
What is the basis of product
heteroduplex translocation?
Single
Nucleotide
Addition
Cycle
Substrate complex
Product complex
Translocation
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Nucleotide Incorporation Cycle
• Mg-ATP and Mg-Pyrophosphate play key
roles in the fingers conformational change
• Mg-Pyrophosphate dissociation drives
translocation and strand separation
B family DNA polymerases also show
a fingers domain conformational
change and a translocated product
DNA
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How do DNA and
RNA polymerases displace the
non template strand during DNA
or RNA synthesis?
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The DNA strand displacement activity
of phi 29 pol is explained by the
template strand passing through a
tunnel that is too small to
accommodate the non-template strand
that is displaced
Structural basis of strand displacement
Downstream
template
Primer
Upstream Template
duplex
Patel and Picha, 2000.
3 Paths to the active site
dNTP
Upstream
duplex
Upstream
duplex
Downstream
template
Acknowledgement
Whitney Yin
David Jeruzalmi
Graham Cheetham
Acknowledgements
Satwik Kamtekar
Andrea Berman
Jimin Wang
Margarita Salas
J.M. Lazaro
Replication by DNA Polymerase III
Replication Fork
DNA Polymerase III a subunit
Thermus aquaticus
3.0Å Resolution
Rfree = 31%
140 KDa
48% Identity to E. coli
Rfactor = 29%
-C
N-
Scott Bailey and Richard Wing
Overall Architecture
Pyrophosphatase
C-terminal Domain
Palm
beta-Binding Domain
Thumb
Fingers
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0
1220
DNA Polymerase Structures
A
B
C
X
Klenow
RB69
PolIII
Pol ß
“Classic Palm”
A vs B
RMSD 2.6Å (82CA)
DALI Z-score 5.0
Nucleotidyl Transferase
C vs X
RMSD 2.6Å (77CA)
DALI Z-score 4.8
Topology of the PolIIIa Palm Domain
Pol
Nucleotidyl Transferase
PolIIIa
Nucleotidyl Transferase
RB69
Classic Palm
“Classic” vs Nucleotidyl Transferase
5’
5’
5’
3’
3’
Classic Palm
Duplex
DNA
5’
3’
3’
Nucleotidyl Transferase
Question
If the last common ancestor of Bacteria and
Eukaryotes had a DNA genome, why are replicative
DNA polymerases not homologous?
Bacteria
Archaeal
Eukaryotes
Last Common Ancestor (LCA)
Speculations
Several Models, but all fall into 3 broad categories:
1. The LCA did not use DNA as its genetic material
2. Both “Classic” and Nucleotidy Transferase domains where utilized
3. DNA Replication was performed by machine X
Bacteria
Archaeal
Eukaryotes
Last Common Ancestor
(LCA)
Speculations
3. DNA Replication was performed by machine X
Machine X:
May have been a ribozyme DNA Polymerase
Originating in the “RNA World”
Replaced domain by domain in each lineage
Bacteria
Archaeal
Eukaryotes
Last Common Ancestor
(LCA)
DnaB Helicase: Overall Fold
Scott Bailey
Richard Wing
N-terminal
Domain
Linker region
C-terminal
RecA Domain
How the CCA-adding Enzyme Adds CCA to
Immature tRNA Without Using an
Oligonucleotide Template: A Story with a
Good End
end
The universally conserved tRNA CCAC CA
• Aminoacylation site -- amino acid attachment site
• Interacts with the 50S ribosome for protein synthesis
Problem--The 3’ CCA of tRNA is:
NOT encoded in many eubacterial and archaeal tRNA genes
NOT encoded in nearly all eukaryotic tRNA genes
C
73
74
CC
CC A
74 75
74 75 76
CTP
CTP
ATP
CCA-adding
enzyme
CCA-adding
enzyme
CCA-adding
enzyme
Solution--The tRNA CCA-adding enzymes
• Catalyze post-transcriptional addition of CCA using CTP/ATP
• A ~50kD polymerase working in a template-independent mode
How does the CCA-adding enzyme
add CCA without using a nucleic
acid template?
Multiple binding site models
• Single subunit, multiple sites
• Multiple subunit, single site/subunit (Scrunching-shuttling Model)
Understanding the structural basis of a biological
process requires structures of the assembly that
carries out the process at each step in the process.
Substrate Constructs
Duplex RNAs that mimic the acceptor stem of tRNA
Step2, ending with C74:
Step3, ending with C75:
Termination, full-length Yeast tRNAphe
Density for AfCCA-tRNA Complex
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Comparison with tRNA complex
ACC75 shown in blue
AC74 +CTP
ACC75 +ATP
tRNA product
How does the enzyme select ATP and
CTP (not UTP or GTP) as substrate?
Nucleotide binding pocket
Hydrogen-bonding complementarity excludes UTP and GTP
Why does AfCCA add CTP to
C74 and not ATP and ATP to C75
and not CTP?
Nucleotide binding pocket
Size and shape of the binding pocket distinguish between CTP and ATP
A flexible head domain
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Summary
Class I
• A single binding pocket made from both protein and tRNA
• Sequence specificity:
i) Hydrogen-bonding complementarity excludes UTP and GTP.
ii) Size and shape of the binding pocket discriminate between
CTP or ATP.
• Length specificity (Termination):
Stacked conformation of the CCA-end and limited size of the
binding pocket.
Acknowledgements
Class I CCA-adding enzyme and
substrate complexes
Yong Xiong
Class II CCA-adding enzyme
Fang Li
Yong Xiong
JiminWang
Alan Weiner
HyunDae Cho
A New Spin in DNA Recombination:
Structural Studies of the
Resolvase/Invertase Family
gd transposition
Site-specific Recombination
by wild-type gd-resolvase
Pre-synaptic
Complex
Resolution
Synapsis
Formation
Dissociation
-
2-3’
-
-
Partial
Synaptosome
180o
-
-
+
-
Synaptic
Complex
Crossover site cleavage and exchange
res: 114 bp recombination site
I
II
III
28
34
25
bp
Enz-Ser-ÖH
Strand exchange
Enz-Ser-O
Phospho-serine linked
covalent intermediate
180º
Rice and Steitz, 1994
Rotate WY
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Activated mutants from Glasgow
Martin Boocock, Marshall Stark, et al.
Recombines site I
substrates (that lack
sites II and III)
Gary Sarkis added Arg2Ala
And Glu56Lys to gamma delta
Resolvase clone
Gly 101 Ser
Glu 102 Tyr
Met 103 Ile
“SYI”
50Å
Tetramer covalently linked to 4 cleaved DNAs
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Pre-synaptic
Complex
S10
Yang and Steitz, Cell, 1995
Synaptic
Complex
Tertiary Structure Change
Conformational Change in one Subunit
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Quaternary Structure Change
Interface Change -- Flat Interface Created
How Flat is the Interface?
Survey of 1029 Unique Protein Interfaces
(SPIN-PP, Hitz & Honig)
areaimol, geomcalc (CCP4)
Resolvase Synaptic Interface
Variation of Surface Area During Subunit Rotation
Area per anti-parallel dimer
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2
Variation: 900 Å to 1200Å
2
~ 6-9 Kcal/mol Difference
2
(100 Å
~ 2-3 Kcal/mol )
Free Energy during Rotation?
Backbone Restrained; van der
Waals Repulsion of Side Chains
Close-up View of Side Chains During Subunit Rotation
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Linking subunits with disulfide bonds
V114C
A74C
Crosslinks across the
Flat interface prevents strand exchange
But not strand cleavage.
The DNAs to be recombined lie
on the outside of a synaptic
resolvase tetramer and appear to
be recombined through subunit
rotation
Acknowledgements
Weikai Li
Nigel D. F. Grindley
Satwik Kametar
Gary J. Sarkis
Yong Xiong
NSLS, CHESS,
APS Staff
Why is peptidyl-tRNA not
hydrolyzed in the absence of A-site
tRNA?
The DNA in the binary structure is post-translocation
templating base
insertion site
Phi29 binary complex (primer/template)
Phi 29 ternary complex (2’3’-dideoxy-terminated primer/template + incoming dNTP)
Toward a mechanism of translocation in Phi29 DNA polymerase (Pol II family)
open
Fingers
subdomain
Phi29 binary complex
Phi 29 ternary complex
closed
Incoming dNTP
Templating base
Pivot point
<2Å
Catalytic carboxylates
Steric gate residue
Crick’s central dogma of molecular biology:
DNA makes DNA makes RNA makes protein