Transcript Enzyme Mechanisms - Illinois Institute of Technology

Nucleic Acid
Structure
Andy Howard
Introductory Biochemistry
7 October 2008
Biochemistry: Nucleic Acid
Chem&Struct
10/02/08
What we’ll discuss
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Small RNAs
DNA & RNA
Hydrolysis
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RNA, DNA
Restriction enzymes
DNA sequencing
DNA secondary
structure: A, B, Z
Folding kinetics
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Supercoils
Nucleosomes
Chromatin and
chromosomes
Lab synthesis of genes
tRNA & rRNA structure
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Other small RNAs
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21-28 nucleotides
Target RNA or DNA through
complementary base-pairing
Several types, based on function:
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Small interfering RNAs (q.v.)
microRNA: control developmental timing
Small nucleolar RNA: catalysts that (among
other things) create the oddball bases
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
snoRNA77
courtesy Wikipedia
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siRNAs and gene
silencing
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QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Small interfering RNAs block specific
protein production by base-pairing to
complementary seqs of mRNA to form
dsRNA
DS regions get degraded & removed
This is a form of gene silencing or RNA
interference
RNAi also changes chromatin structure
and has long-range influences on
expression
10/02/08 Biochemistry: Nucleic Acid Chem&Struct
Viral p19
protein
complexed to
human 19-base
siRNA
PDB 1R9F
1.95Å
17kDa protein
p. 4 of 38
Do the differences between
RNA and DNA matter? Yes!
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DNA has deoxythymidine, RNA has uridine:
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cytidine spontaneously degrades to uridine
dC spontaneously degrades to dU
The only dU found in DNA is there because of
degradation: dT goes with dA
So when a cell finds dU in its DNA, it knows it
should replace it with dC or else synthesize dG
opposite the dU instead of dA
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Ribose vs. deoxyribose
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Presence of -OH on 2’ position makes the 3’
position in RNA more susceptible to
nonenzymatic cleavage than the 3’ in DNA
The ribose vs. deoxyribose distinction also
influences enzymatic degradation of nucleic
acids
I can carry DNA in my shirt pocket, but not
RNA
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Backbone hydrolysis of
nucleic acids in base
(fig. 10.29)
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Nonenzymatic hydrolysis in base occurs
with RNA but not DNA, as just mentioned
Reason: in base, RNA can form a specific
5-membered cyclic structure involving
both 3’ and 2’ oxygens
When this reopens, the backbone is
cleaved and you’re left with a mixture of
2’- and 3’-NMPs
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Enzymatic cleavage of oligoand polynucleotides
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Enzymes are phosphodiesterases
Could happen on either side of the P
3’ cleavage is a-site; 5’ is b-site.
Endonucleases cleave somewhere on
the interior of an oligo- or polynucleotide
Exonucleases cleave off the terminal
nucleotide
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An a-specific
exonuclease
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A b-specific
exonuclease
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Specificity in nucleases
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Some cleave only RNA, others only DNA,
some both
Often a preference for a specific base or
even a particular 4-8 nucleotide
sequence (restriction endonucleases)
These can be used as lab tools, but they
evolved for internal reasons
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Variety of nucleases
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Restriction endonucleases
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Evolve in bacteria as antiviral tools
“Restriction” because they restrict the
incorporation of foreign DNA into the bacterial
chromosome
Recognize and bind to specific palindromic DNA
sequences and cleave them
Self-cleavage avoided by methylation
Types I, II, III: II is most important
I and III have inherent methylase activity; II has
methylase activity in an attendant enzyme
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What do we mean by
palindromic?
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In ordinary language, it means a phrase
that reads the same forward and back:
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Madam, I’m Adam. (Genesis 3:20)
Eve, man, am Eve.
Able was I ere I saw Elba. (Napoleon)
A man, a plan, a canal: Panama!
(T. Roosevelt)
With DNA it means the double-stranded
sequence is identical on both strands
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Quirky math question to ponder
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Numbers can be palindromic:
484, 1331, 727, 595…
Some numbers that are palindromic have
squares that are palindromic…
222 = 484, 1212 = 14641, . . .
Question: if a number is perfect square
and a palindrome, is its square root a
palindrome? (answer will be given orally)
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Palindromic DNA
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G-A-A-T-T-C
Single strand isn’t symmetric: but the
combination with the complementary
strand is:
G-A-A-T-T-C
C-T-T-A-A-G
These kinds of sequences are the
recognition sites for restriction
endonucleases. This particular
hexanucleotide is the recognition
sequence for EcoRI.
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Cleavage by restriction
endonucleases
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Breaks can be cohesive (if they’re off-center
within the sequence) or non-cohesive (blunt) (if
they’re at the center)
EcoRI leaves staggered 5’-termini: cleaves
between initial G and A
PstI cleaves CTGCAG between A and G, so it
leaves staggered 3’-termini
BalI cleaves TGGCCA in the middle: blunt!
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iClicker question:
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Which of the following is a potential
restriction site?
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(a) ACTTCA
(b) AGCGCT
(c) TGGCCT
(d) AACCGG
(e) none of the above.
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Example for EcoRI
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5’-N-N-N-N-G-A-A-T-T-C-N-N-N-N-3’
3’-N-N-N-N-C-T-T-A-A-G-N-N-N-N-5’
Cleaves G-A on top, A-G on bottom:
5’-N-N-N-N-GA-A-T-T-C-N-N-N-N-3’
3’-N-N-N-N-C-T-T-A-AG-N-N-N-N-5’
Protruding 5’ ends:
5’-N-N-N-N-G
A-A-T-T-C-N-N-N-N-3’
3’-N-N-N-N-C-T-T-A-A
G-N-N-N-N-5’
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How often?
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4 types of bases
So a recognition site that is 4 bases long
will occur once every 44 = 256 bases on
either strand, on average
6-base site: every 46= 4096 bases, which
is roughly one gene’s worth
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EcoRI
structure
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Dimeric structure
enables recognition of
palindromic sequence
 sandwich in each
monomer
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
EcoRI pre-recognition
complex
PDB 1CL8
57 kDa dimer + DNA
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Methylases
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QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
A typical bacterium protects
its own DNA against
HhaI methyltransferase
cleavage by its restriction
PDB 1SVU
endonucleases by
2.66Å; 72 kDa dimer
methylating a base in the
restriction site
Methylating agent is
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
generally Sadenosylmethionine
Structure
10/02/08 Biochemistry: Nucleic Acid Chem&Struct
courtesy
steve.gb.com
p. 22 of 38
Use of restriction enzymes
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Nature made these to protect bacteria; we use
them to cleave DNA in analyzable ways
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Similar to proteolytic digestion of proteins
Having a variety of nucleases means we can get
fragments in multiple ways
We can amplify our DNA first
Can also be used in synthesis of inserts that we
can incorporate into plasmids that enable us to
make appropriate DNA molecules in bacteria
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Sanger dideoxy method
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Incorporates DNA replication as an analytical
tool for determining sequence
Uses short primer that attaches to the 3’ end of
the ssDNA, after which a specially engineered
DNA polymerase
Each vial includes one dideoxyXTP and 3
ordinary dXTPs; the dideoxyXTP will be
incorporated but will halt synthesis because the
3’ position is blocked.
See figs. 11.3 & 11.4 for how these are read out
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Automating dideoxy
sequencing
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Laser fluorescence detection allows for
primer identification in real time
An automated sequencing machine can
handle 4500 bases/hour
That’s one of the technologies that has
made large-scale sequencing projects
like the human genome project possible
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DNA secondary structures
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If double-stranded DNA were simply a straightlegged ladder:
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Base pairs would be 0.6 nm apart
Watson-Crick base-pairs have very uniform
dimensions because the H-bonds are fixed lengths
But water could get to the apolar bases
So, in fact, the ladder gets twisted into a helix.
The most common helix is B-DNA, but there are
others. B-DNA’s properties include:
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Sugar-sugar distance is still 0.6 nm
Helix repeats itself every 3.4 nm, i.e. 10 bp
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Properties of B-DNA
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Spacing between base-pairs along helix
axis = 0.34 nm
10 base-pairs per full turn
So: 3.4 nm per full turn is pitch length
Major and minor grooves, as discussed
earlier
Base-pair plane is almost perpendicular
to helix axis
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Major groove in B-DNA
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H-bond between adenine NH2 and
thymine ring C=O
H-bond between cytosine amine and
guanine ring C=O
Wide, not very deep
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Minor groove in B-DNA
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H-bond between adenine ring N and
thymine ring NH
H-bond between guanine amine and
cytosine ring C=O
Narrow but deep
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Cartoon
of AT
pair in
B-DNA
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Cartoon
of CG
pair in
B-DNA
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What holds duplex
B-DNA together?
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H-bonds (but just barely)
Electrostatics: Mg2+  –PO4-2
van der Waals interactions
 - interactions in bases
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Solvent exclusion
Recognize role of grooves in defining
DNA-protein interactions
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Helical twist
(fig. 11.9a)
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Rotation about the
backbone axis
Successive base-pairs
rotated with respect to
each other by ~ 32º
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Propeller
twist
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Improves overlap of
hydrophobic surfaces
Makes it harder for
water to contact the
less hydrophilic parts
of the molecule
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A-DNA (figs. 11.10)
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In low humidity this forms naturally
Not likely in cellular duplex DNA, but it does form
in duplex RNA and DNA-RNA hybrids because
the 2’-OH gets in the way of B-RNA
Broader
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2.46 nm per full turn
11 bp to complete a turn
Base-pairs are not perpendicular to helix axis:
tilted 19º from perpendicular
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Z-DNA (figs. 11.10)
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Forms in alternating Py-Pu sequences
and occasionally in PyPuPuPyPyPu,
especially if C’s are methylated
Left-handed helix rather than right
Bases zigzag across the groove
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Getting from B to Z
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Can be accomplished without breaking
bonds
… even though purines have their
glycosidic bonds flipped (anti -> syn) and
the pyrimidines are flipped altogether!
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DNA is dynamic
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Don’t think of these diagrams as static
The H-bonds stretch and the torsions
allow some rotations, so the ropes can
form roughly spherical shapes when not
constrained by histones
Shape is sequence-dependent, which
influences protein-DNA interactions
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