Transcript Nucleic Acids: RNA and chemistry

Nucleic Acids:
DNA, RNA and chemistry
Andy Howard
Introductory Biochemistry
7 October 2010
Biochemistry:Nucleic Acids II
10/07/2010
DNA & RNA
structure & function
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DNA and RNA are dynamic molecules,
but understanding their structural realities
helps us understand how they work
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What we’ll discuss
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DNA structure
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Characterizations
B, A, and Z-DNA
Dynamics
Function
RNA:
structure & types
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DNA & RNA
Hydrolysis
alkaline
 RNA, DNA
nucleases
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mRNA
tRNA
rRNA
Small RNAs
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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
From Molecular
perpendicular to helix axis
Biology web-book
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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
From Berg et al.,
Biochemistry
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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 basepairs 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 &
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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Summaries of A, B, Z DNA
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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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What does DNA do?
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Serve as the storehouse and the propagator of
genetic information:
That means that it’s made up of genes
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Some code for mRNAs that code for protein
Others code for other types of RNA
Genes contain non-coding segments (introns)
But it also contains stretches that are not parts of
genes at all and are serving controlling or
structural roles
Avoid the term junk DNA!
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Ribonucleic acid
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We’re done with DNA for the moment.
Let’s discuss RNA.
RNA is generally, but not always, singlestranded
The regions where localized base-pairing
occurs (local double-stranded regions)
often are of functional significance
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RNA physics & chemistry
RNA molecules vary widely in size, from a few bases in
length up to 10000s of bases
 There are several types of RNA found in cells
Type
% %turnSize, Partly Role
RNA
over
bases DS?
mRNA
3
25
50-104 no
protein template
tRNA
15
21
55-90 yes
aa activation
rRNA
80
50
102-104 no
transl. catalysis &
scaffolding
sRNA
2
4
15-103 ?
various
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Messenger RNA
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mRNA: transcription vehicle
DNA 5’-dAdCdCdGdTdAdTdG-3’
RNA 3’- U G G C A U A C-5’
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typical protein is ~500 amino acids;
3 mRNA bases/aa: 1500 bases (after splicing)
Additional noncoding regions (see later) brings it
up to ~4000 bases =
4000*300Da/base=1,200,000 Da
Only about 3% of cellular RNA but instable!
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Relative quantities
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Note that we said there wasn’t much
mRNA around at any given moment
The amount synthesized is much
greater because it has a much shorter
lifetime than the others
Ribonucleases act more avidly on it
We need a mechanism for eliminating it
because the cell wants to control
concentrations of specific proteins
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mRNA processing in Eukaryotes
Genomic DNA
Unmodified mRNA produced therefrom
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# bases (unmodified mRNA) =
# base-pairs of DNA in the gene…
because that’s how transcription works
BUT the number of bases in the unmodified
mRNA > # bases in the final mRNA that actually
codes for a protein
SO there needs to be a process for getting rid of
the unwanted bases in the mRNA: that’s what
splicing is!
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Splicing: quick summary
Genomic DNA
transcription
Unmodified mRNA produced therefrom
exon
intron
exon
intron
exon
intron
splicing
exon
exon
(Mature transcript)
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exon
translation
Typically the initial eukaryotic message
contains roughly twice as many bases as the
final processed message
Spliceosome is the nuclear machine
(snRNAs + protein) in which the introns are
removed and the exons are spliced together
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Heterogeneity via
spliceosomal flexibility
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Specific RNA sequences in the initial
mRNA signal where to start and stop
each intron, but with some flexibility
That flexibility enables a single gene to
code for multiple mature RNAs and
therefore multiple proteins
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Transfer RNA
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tRNA: tool for engineering protein
synthesis at the ribosome
Each type of amino acid has its
own tRNA, responsible for
positioning the correct aa into the
growing protein
Roughly T-shaped or Y-shaped
molecules; generally 55-90 bases
long
15% of cellular RNA
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Phe tRNA
PDB 1EVV
76 bases
yeast
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Secondary and Tertiary
Structure of tRNA
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Extensive H-bonding creates four double
helical domains, three capped by loops, one
by a stem
Only one tRNA structure (alone) is known
Phenylalanine tRNA is "L-shaped"
Many non-canonical bases found in tRNA
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tRNA
structure:
overview
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Amino acid
linkage to
acceptor stem
Amino acids are linked to the 3'-OH
end of tRNA molecules by an
ester bond formed between the
carboxyl group of the amino acid
and the 3'-OH of the terminal
ribose of the tRNA.
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Yeast phetRNA
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Note
nonstandard
bases and
cloverleaf
structure
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Ribosomal RNA
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rRNA: catalyic and scaffolding
functions within the ribosome
Responsible for ligation of new
amino acid (carried by tRNA)
onto growing protein chain
Can be large: mostly 500-3000
bases
a few are smaller (150 bases)
Very abundant: 80% of cellular
RNA
Relatively slow turnover
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23S rRNA
PDB 1FFZ
602 bases
Haloarcula
marismortui
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Small RNA
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sRNA: few bases / molecule
often found in nucleus; thus it’s often
called small nuclear RNA, snRNA
Involved in various functions, including
processing of mRNA in the spliceosome
Protein Prp31
Some are catalytic
complexed to U4
Typically 20-1000 bases
snRNA
Not terribly plentiful: ~2 % of total RNA
PDB 2OZB
33 bases +
85kDa
heterotetramer
Human
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iClicker quiz
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1. Shown is the lactim
form of which nucleic
acid base?
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Uracil
Guanine
Adenine
Thymine
None of the above
HN
O
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N
OH
lactim
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iClicker quiz #2
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Suppose someone reports that he has
characterized the genomic DNA of an
organism as having 29% A and 22% T. How
would you respond?
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(a) That’s a reasonable result
(b) This result is unlikely because [A] ~ [T] in
duplex DNA
(c) That’s plausible if it’s a bacterium, but not if
it’s a eukaryote
(d) none of the above
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Unusual bases in RNA
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mRNA, sRNA mostly ACGU
rRNA, tRNA have some odd ones
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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
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Viral p19
protein
complexed to
human 19-base
siRNA
PDB 1R9F
1.95Å
17kDa protein
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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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Why alkaline hydrolysis works
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Cyclic phosphate intermediate stabilizes
cleavage product
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The cyclic intermediate
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Hydroxyl or water
can attack fivemembered Pcontaining ring on
either side and
leave the –OP on
2’ or on 3’.
O
H
N
O
O
O-
O
P
N
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O
OO
O
P
O-
O
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Consequences
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So RNA is considerably less stable
compared to DNA, owing to the formation
of this cyclic phosphate intermediate
DNA can’t form this because it doesn’t
have a 2’ hydroxyl
In fact, deoxyribose has no free
hydroxyls!
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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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Enzymatic RNA
hydrolysis
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Ribonucleases operate through
a similar 5-membered ring
intermediate: see fig. 19.29 for
bovine RNAse A:
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His-119 donates proton to 3’-OP
His-12 accepts proton from 2’-OH
Cyclic intermediate forms with
cleavage below the phosphate
Ring collapses, His-12 returns
proton to 2’-OH, bases restored
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PDB
1KF8
13.6 kDa
monomer
bovine
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Variety of nucleases
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