Transcript Enzyme Mechanisms - Illinois Institute of Technology

Nucleic Acid
Chemistry & Structure
Andy Howard
Introductory Biochemistry
2 October 2008
Biochemistry: Nucleic Acid
Chem&Struct
10/02/08
What we’ll discuss
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Syn, anti revisited
Nucleotides
Oligo- and polynucleotides
DNA duplexes and helicity
RNA: structure & types
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Syn versus anti
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Monophosphorylated
nucleosides
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NH2
N
N
N
We have specialized names for HO
the 5’-phospho derivatives of the
nucleosides, i.e. the nucleoside
monophosphates:
They are nucleotides
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N
O
OO
OP
HO
O
adenylate
Adenosine 5’-monophosphate =
AMP = adenylate
GMP = guanylate
CMP = cytidylate
UMP = uridylate
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pKa’s for base N’s and PO4’s
Nucleotide pKa base-N pK1 of PO4 pK2 of PO4
5’-AMP
3.8(N-1)
0.9
6.1
5’-GMP
9.4 (N-1)
0.7
6.1
2.4 (N-7)
5’-CMP
4.5 (N-3)
0.8
6.3
5’-UMP
9.5 (N-3)
1.0
6.4
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UV absorbance
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These aromatic rings absorb around
260
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Deoxynucleotides
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O
Similar nomenclature
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dAMP =
deoxyadenylate
dGMP =
deoxyguanylate
dCMP =
deoxycytidylate
dTTP (= TTP) =
deoxythymidylate =
thymidylate
N
HN
H2N
N
N
O
OO
OP
HO
O
deoxyguanylate
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Cyclic
phosphodiesters
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3’ and 5’ hydroxyls are both involved
in -O-P-O bonds, forming a 6-membered ring
(-C5’-C4’-C3’-O-P-O-)
cAMP and cGMP are the important ones
(see previous lecture!)
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Di- and triphosphates
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Phosphoanhydride bonds link second and
perhaps third phosphates to the 5’-OH on
the ribose moiety
O
N
O
H2N
O
O
O
P
P
P
O
N
O
O-
O
O-
OH
O-
Mg2+
OH
HO
cytidine triphosphate
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These are polyprotic acids
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They can dissociate 3 protons (XDP) or 4
protons (XTP) from their phosphoric acid
groups
The ionized forms are frequently associated
with divalent cations (Mg2+, Mn2+, others)
The -O-P-O bonds beyond the first one are
actually phosphoric anhydride linkages
Phosphoanhydrides are acid-labile: quantitative
liberation of Pi in 1N HCl for 7 minutes @100ºC
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NTPs: carriers of chemical
energy
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ATP is the energy currency
GTP is important in protein synthesis
CTP used in phospholipid synthesis
UTP forms activated intermediates with
sugars (e.g. UDP-glucose)
… and, of course, they’re substrates to
build up RNA and DNA
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Bases are information
symbols
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Base and sugar aren’t directly involved in
metabolic roles of the XTPs
But different XTPs do different things, so
there are recognition components to the
relevant enzymatic systems that notice
whether X is A, U, C, or G
Even in polynucleotides the bases play
an informational role
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Oligomers and Polymers
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Monomers are nucleotides or
deoxynucleotides
Linkages are phosphodiester linkages
between 3’ of one ribose and 5’ of the next
ribose
It’s logical to start from the 5’ end for
synthetic reasons
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Typical DNA dinucleotide
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Various notations: this is pdApdCp
Leave out the p’s if there’s a lot of them!
-O
OP
O
O
O
-O
N
O-
N
P
O
O
O
O
N
-O
P
O
HN
O
NH2
O
N
O
N
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NH2
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DNA structure
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Many years of careful
experimental work enabled
fabrication of double-helical
model of double-stranded
DNA
Explained [A]=[T], [C]=[G]
Specific H-bonds stabilize
double-helical structure:
see fig. 10.20
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What does double-stranded
DNA really look like?
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Picture on previous slide emphasizes
only the H-bond interactions; it ignores
the orientation of the sugars, which are
actually tilted relative to the helix axis
Planes of the bases are almost
perpendicular to the helical axes on both
sides of the double helix
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Sizes (cf fig. 10.20, 11.7)
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Diameter of the double helix: 2.37nm
Length along one full turn:
10.4 base pairs = pitch = 3.40nm
Distance between stacked base pairs =
rise = 0.33 nm
Major groove is wider and shallower;
minor groove is narrower and deeper
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What stabilizes this?
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Variety of stabilizing
interactions
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Stacking of base pairs
Hydrogen bonding between
base pairs
Hydrophobic effects (burying
bases, which are less polar)
Charge-charge interactions:
phosphates with Mg2+ and
cationic proteins
Courtesy
dnareplication.info
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How close to instability is it?
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Pretty close.
Heating DNA makes it melt: fig. 11.14
The more GC pairs, the harder it is to
melt
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Weaker stacking interactions in A-T
One more H-bond per GC than per AT
We’ll get into DNA structure a lot more
later in this lecture
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iClicker quiz
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1. What positions of a pair of
aromatic rings leads to stabilizing
interactions?
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(a) Parallel to one another
(b) Perpendicular to one another
(c) At a 45º angle to one another
(d) Both (a) and (b)
(e) All three: (a), (b), and ( c)
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Second iClicker question
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2. Which has the highest molecular
mass among the compounds listed?
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(a) cytidylate
(b) thymidylate
(c) adenylate
(d) adenosine triphosphate
(e) they’re all the same MW
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Base composition for DNA
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As noted, [A]=[T], [C]=[G] because of
base pairing
[A]/[C] etc. not governed by base pairing
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Can vary considerably (table 10.3)
E.coli : [A], [C] about equal
Mycobacterium tuberculosis: [C] > 2*[A]
Mammals: [C] < 0.74*[A]
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Molar ratios for various
organisms’ DNA (table 10.3)
Source
Ox
Human
Hen
Salmon
Wheat
Yeast
A/G
1.29
1.56
1.45
1.43
1.22
1.67
H.influenzae 1.74
E.coli K-12 1.05
B. schatz
0.7
T/C
1.43
1.75
1.29
1.43
1.18
1.92
1.54
0.95
0.6
A/T
1.04
1.0
1.06
1.02
1.00
1.03
1.07
1.09
1.12
G/C
1.00
1.0
0.91
1.02
0.97
1.20
0.91
0.99
0.89
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Pur/Pyr
1.1
1.0
0.99
1.02
0.99
1.0
1.0
1.0
1.0
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What did this mean in 1950?
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[A]=[T] and [C]=[G] suggested that if the
molecule involved two strands, there
should be complementarity between
them, i.e., if there’s an A on one strand,
there will be a T on the other one
Unfortunately it wasn’t entirely clear that
the molecule was two-stranded!
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The Watson-Crick
contribution
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Interpreting the X-ray fiber
diffraction photographs taken by
Rosalind Franklin and Maurice
Wilkins, W&C built a ball-andstick model for a two-stranded
form of DNA
They were able to show that
their model was consistent with
Franklin’s data
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QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
p. 25 of 43
So how is DNA organized?
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Linear sequence is simple to describe:
Two strands, each very long and
containing 105 - 108 bases
Each base has a complementary base on
the other strand
Specific hydrogen bonding patterns
define the complementarity
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Higher levels of organization
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Just as with protein tertiary structure, DNA
structure has higher levels beyond the basepairing, beginning with coiling into a double helix
Eukaryotes:
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Organization of double helix into loop structures
of ~200 base pairs coiled around a protein
complex called the histone octamer
Further organization of those loops into larger
structures culminating in formation of
chromosomes
Prokaryotes: similar but simpler higher-level
structures culminating in (often circular)
chromosomes
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Supercoiling
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Refers to levels of organization of DNA
beyond the immediate double-helix
We describe circular DNA as relaxed if
the closed double helix could lie flat
It’s underwound or overwound if the ends
are broken, twisted, and rejoined.
Supercoils restore 10.4 bp/turn relation
upon rejoining
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Supercoiling
and flat DNA
Diagram courtesy SIU Carbondale
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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
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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
% %turnRNA over
mRNA
3
25
tRNA
15
21
rRNA
80
50
Size,
bases
50-104
55-94
102-104
Hbond
stabil.?
no
yes
yes
sRNA
12-200
yes
2
4
Role
in translation
protein template
aa activation
transl. catalysis
& scaffolding
various
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Unusual bases in RNA
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mRNA, sRNA mostly A,C,G,U
rRNA, tRNA have some odd ones
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Messenger RNA
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Contains the codons that define protein
sequence
Each codon (3 bases) codes for 1 amino acid
Synthesized during transcription, like all other
types of RNA
Relatively small % of RNA mass in the cell; but
short-lived, so:
Higher % of RNA synthesis devoted to mRNA
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Prokaryotic mRNA
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One mRNA with a single promoter will
contain coding information for several
proteins, i.e., 1 promoter, several genes
Defined stop codons show the ribosome
where to put in the breaks
Translation closely coupled to
transcription, unlike eukaryotic systems,
where they’re separated in space & time
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Eukaryotic mRNA
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One mRNA per protein
But the mRNA will be initially synthesized with
noncoding segments (introns) interspersed
between the coding segments (exons):
heterogeneous nuclear RNA, hnRNA
snRNPs (q.v.) in nucleus splice out the introns,
tying together the exons to make the mature
transcript
Each mRNA will end with a poly(A) tail, added
after transcription
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Ribosomes and rRNA
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Ribosome is 65% RNA, rest protein
Lots of intrastrand H-bonds
Ribosomes characterized by
sedimentation coefficients
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E.coli: 50S piece+30S piece  70S total
Eukaryotes 60S + 40S  80S total
rRNA has pseudouridine, ribothymidine,
methylated bases
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Prokaryotic
ribosomes
(fig.10.25a)
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Eukaryotic
ribosomes
(fig. 10.25b)
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Transfer RNA
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QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Each tRNA carries a specific
amino acid to the ribosomal
protein synthesis machine
One full set of tRNA at each
cellular site of protein synthesis
(cytoplasm, mitochondrion,
chloroplast)
These are small molecules: 55-94
bases
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A/T site
tRNA model
based on
cryoEM
complex
PDB 1QZA
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tRNA contents
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Many modified bases
CCA on the 3’-end is
attached to the amino acid
Catalytic attachment of
amino acid to protein is
catalyzed by an adenine in
one of the 50S rRNAs
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Qui ckT ime™ and a
T IFF (Uncompres sed) dec ompres sor
are needed to s ee this pic ture.
Dieter Söll
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Small nuclear
RNAs
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QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
snRNA found mostly
in nucleus
100-200 nucleotides
Image courtesy
closely associated with proteins
Richard Lührmann,
& with other RNA molecules
Göttingen
Mostly in ribonucleoprotein particles
(snRNPs), which are involved in mRNA
processing, converting full-length transcript
into smaller transcript in which introns have
been removed, leaving only the exons
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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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iClicker question 3
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Suppose you isolate an RNA molecule
that consists of 1500 bases. It is
probably:
(a) tRNA
(b) mRNA
(c) rRNA
(d) either mRNA or rRNA
(e) none of the above.
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