Transcript Nucleic Acids: RNA and chemistry

Nucleic Acids:
RNA and chemistry
Andy Howard
Introductory Biochemistry
13 October 2009
Biochemistry:Nucleic Acids II
10/13/2009
What we’ll discuss
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RNA: structure & types
 mRNA
 tRNA
 rRNA
 Small RNAs
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DNA & RNA Hydrolysis
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alkaline
RNA, DNA nucleases
Restriction enzymes
DNA & RNA dynamics
and density
measurements
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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
30-103 ?
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 base pairs 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 alatRNA
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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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Ribosomal composition
(fig.10.22)
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Bacterial ribosome
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30S subunit: 16S RNA + 21 proteins
50S subunit:
23S RNA + 5S RNA + 31 proteins
Eukaryotic ribosome
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40S subunit: 18S RNA + 33 proteins
60S subunit:
(28S+5.85S) RNA, 5S RNA + 49 proteins
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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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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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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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How many varieties of each
class?
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mRNA: thousands
(one per protein transcript)
tRNA: one per codon plus a few more
rRNA: a few per organism—
see rRNA slide
sRNA: dozens (?)
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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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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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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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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.
Sex at noon taxes.
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
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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 3:
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3. 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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QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Dimeric structure
enables recognition of
palindromic sequence
 sandwich in each
monomer
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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
courtesy
steve.gb.com
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The biology problem
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How does the bacterium mark its own DNA
so that it does replicate its own DNA but not
the foreign DNA?
Answer: by methylating specific bases in its
DNA prior to replication
Unmethylated DNA from foreign source
gets cleaved by restriction endonuclease
Only the methylated DNA survives to be
replicated
Most methylations are of A & G,
but sometimes C gets it too
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How this works
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When an unmethylated specific
sequence appears in the DNA, the
enzyme cleaves it
When the corresponding methylated
sequence appears, it doesn’t get cleaved
and remains available for replication
The restriction endonucleases only bind
to palindromic sequences
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