7/31/2019 223 Course1
1/32
Molecular Genetics-223Spring 2012
nature of the genetic code
maintenance of genes through DNA replication
transcription of information from DNA to mRNA
translation of mRNA into protein
DNA repair
Recombination
Intron splicing
7/31/2019 223 Course1
2/32
Brenner S., Jacob F., and Meselson M. (1961)
An unstable intermediate carrying information fromgenes to ribosomes for protein synthesis.
Nature 190: 576-581 (13 May 1961)
7/31/2019 223 Course1
3/32
RNA
Composition
StructureFunction
Stability
7/31/2019 223 Course1
4/32
7/31/2019 223 Course1
5/32
7/31/2019 223 Course1
6/32
7/31/2019 223 Course1
7/32
7/31/2019 223 Course1
8/32
Chemistry of Information
N
NNH
N
NH2
HN
NNH
N
O
H2N
N
NH
NH2
O
NH
NH
O
O
adenine (A) guanine (G) cytosine (C) uracil (U)
Nucleoside bases found in RNA:
Nucleic acids are polymers ofnucleotides.Each nucleotide includes a nucleoside base
that is either
a purine (adenine or guanine), or
a pyrimidine (cytosine, uracil, or thymine).
7/31/2019 223 Course1
9/32
RNA species may contain modified bases. Examples:
N
NNH
N
NH2
HN
NNH
N
O
H2N
N
NH
NH2
O
NH
NH
O
O
adenine (A) guanine (G) cytosine (C) uracil (U)
N
NNH
N
NH2
H3C
+HN
NNH
N
O
H2N
CH3
+
N
NH
NH2
O
CH3+NH
NH
HN
O
O
1-methyladenine (m1A) 7-methylguanine (m7G) 3-methylcytosine (m3C) pseudouracil ()
Nucleoside bases found in RNA:
Examples of modified bases found in tRNA:
7/31/2019 223 Course1
10/32
In a nucleotide, e.g., adenosine monophosphate (AMP),
the base is bound to a ribose sugar, which has a phosphate
in ester linkage to the 5' hydroxyl.
N
NN
N
NH2
adenine adenosine adenosine monophosphate (AMP)
O
OHOH
HH
H
CH2
H
HO
N
NNH
N
NH2
N
NN
N
NH2
O
OHOH
HH
H
CH2
H
OO3P2
ribose
5'
adenine
4'
3' 2'
1'
7/31/2019 223 Course1
11/32
Nucleic acids
have a backbone
of alternating Pi &ribose moieties.
Phosphodiester
linkages form as
the 5' phosphateof one nucleotide
forms an ester link
with the 3' OH of
the adjacent
nucleotide.
A short stretch of
RNA is shown.
N
NN
N
NH2
O
OHO
HH
H
CH2
H
ribose
adenine
P
O
O OO
OHO
HH
H
CH2
H
N
N
NH2
O
P
O
O O
OP
O
O
O
cytosine5'
4'
3' 2'
1'
ribose3'
5'
3'end
5'end
(etc)
nucleic acid
7/31/2019 223 Course1
12/32
Hydrogen bonds link 2 complementary nucleotide bases
on separate nucleic acid strands, or on complementary
portions of the same strand.Conventional base pairs: A & U (or T); C & G.
In the diagram at left, H-bonds are in red. Bond lengths
are inexact. The image is based on X-ray crystallography
of tRNAGln. H atoms are not shown.
N
NNH
N
O
N
N
NH
N
O
H
H
H
H
H
guanine(G)
cytosine(C)
G
C
G C base
pair in tRNA
7/31/2019 223 Course1
13/32
Secondary structure
Base pairing over extended stretches of complementary
base sequences in two nucleic acid strands stabilizes
secondary structure, such as the double helix of DNA.
Stacking interactions between adjacent hydrophobic
bases contribute to stabilization of such secondary
structures. Each base interacts with its neighbors aboveand below, in the ladder-like arrangement of base pairs
in the double helix, e.g., of DNA.
7/31/2019 223 Course1
14/32
Double helical stems arise from base pairing between
complementary stretches of bases within the same strand.
Loops occur where lack of complementarity, or thepresence ofmodified bases, prevents base pairing.
RNA structure:Most RNAs have secondary structure, consisting of
stem & loop domains.
7/31/2019 223 Course1
15/32
The cloverleaf model of
tRNA secondary structureemphasizes the 2 major types
of secondary structure,
stem and loop domains.
tRNAs typically include many modified bases,
particularly in the loop domains.
Tertiary structure depends on interactions of bases at
more distant sites. Many of these interactions involvenon-standard base pairing and/or interactions involving
three or more bases.
tRNAs usually fold into an L-shaped tertiary structure.
anticodon loop
acceptorstemtRNA
7/31/2019 223 Course1
16/32
The amino acid
attaches to the ribose
of the terminal A
(in red) at the 3' end.The anticodon loop is
at the opposite end of
the L shape.
anticodon
acceptor
stem
tRNAPhe
anticodon loop
acceptorstemtRNA
Extending out from the
"acceptor stem", the 3' end of
every tRNA has the sequence CCA.
7/31/2019 223 Course1
17/32
Tertiary base pairs
#46
(m7G)
#22G #13
C
Tertiary base
pairs in tRNAPhe
#46
(m7G)
#22
G
#13
C
Tertiary base
pairs in tRNAPhe
Non-standard H bond interactions, some linking 3 bases,
help stabilize the L-shaped tertiary structure of tRNA. H
atoms are not shown.
7/31/2019 223 Course1
18/32
Some other RNAs,
including viral RNAs &
segments of ribosomalRNAs, fold in
pseudoknots, tertiary
structures that mimic the
3D structure of tRNA.
Pseudoknots are
stabilized by tertiary
(non-standard) H-bondinteractions.
anticodon
acceptorstem
tRNAPhe
7/31/2019 223 Course1
19/32
Aminoacyl-tRNA Synthetases catalyze linkage of theappropriate amino acid to each tRNA. The reaction occurs
in two steps.
In step 1, an O atom of the amino acid a-carboxyl attacks
the P atom of the initial phosphate of ATP.
O
OHOH
HH
H
CH2
H
OPOPOP
O
O
O
O
O O
O
R
H
C C
NH3+
O
O
O
OHOH
HH
H
CH2
H
OPOC
O
O
H
CR
NH2
O
Adenine
Adenine
ATPAmino acid
Aminoacyl-AMP
PPi
7/31/2019 223 Course1
20/32
In step 2, the
2' or 3' OH of
the terminal
adenosine of
tRNA attacks
the amino acidcarbonyl C
atom.
O
OHOH
HH
H
CH2
H
OPOC
O
O
H
CR
NH2
O
Adenine
O
OHO
HH
H
CH2
H
OPO
O
O
Adenine
tRNA
C
HC
O
NH3+
R
tRNA
AMP
Aminoacyl-AMP
Aminoacyl-tRNA
(terminal 3nucleotide
of appropriate tRNA)3 2
7/31/2019 223 Course1
21/32
Aminoacyl-tRNA Synthetase
Summary of the 2-step reaction:
1. amino acid + ATP aminoacyl-AMP + PPi
2. aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP
7/31/2019 223 Course1
22/32
There is a different Aminoacyl-tRNA Synthetase
(aaRS) for each amino acid.
Each aaRS recognizes its particular amino acid and thetRNAs coding for that amino acid.
Domains of tRNA recognized by an
aaRS are called identity elements.
Most identity elements are in theacceptor stem & anticodon loop.
Aminoacyl-tRNA Synthetases arose
early in evolution
anticodon loop
acceptorstemtRNA
7/31/2019 223 Course1
23/32
One of the current models of translation start
7/31/2019 223 Course1
24/32
Key enzymes: 1) Guanylyl transferase,2) Guanine-7-methyltransferase,
3) 2-O-metyltransferase.
7/31/2019 223 Course1
25/32
An example to viral mechanisms
blocking eukaryotic translation
Prevention of cap-dependent translation.
Viral protease (2A-pro) cleaves eIF4G.
7/31/2019 223 Course1
26/32
Firefly luciferase mRNA (with 5 and 3 UTRs from a plant gene)
was electroporated into protoplasts. At intervals, the amount of
luciferase mRNA was checked (to determine half-life), and the
amount of luciferase activity (which reflects the amount of
translation product).
7/31/2019 223 Course1
27/32
Pre-mRNA Polyadenylation
Most cytoplasmic mRNAs have a polyAtail (3 end) of 50-250 Adenylates
Added post-transcriptionally by anenzyme, Poly(A) Polymerase (PAP)
Turns over (recycles) in cytoplasm
y
7/31/2019 223 Course1
28/32
Functions of the PolyA
Tail
1. Promotes mRNA stability- Deadenylation (shortening of the polyA
tail) can trigger rapid degradation of
the mRNA
2. Enhances translation- promotes recruitment by ribosomes
- bound by a polyA-binding protein inthe cytoplasm called PAB1
- synergistic stimulation with Cap!
7/31/2019 223 Course1
29/32
Overview of PolyadenylationMechanism
1. Transcription extendsbeyond mRNA end
2. Transcript is cut at 3 end ofwhat will become themRNA (in green)
3. PolyA Polymerase adds~250 As to 3 end
4. Extra RNA (in red)
degraded
7/31/2019 223 Course1
30/32
Eukaryotic cytoplasmic ribosomes are larger and more
complex than prokaryotic ribosomes.
Ribosome
Source
Whole
Ribosome
Small
Subunit
Large
Subunit
E. coli 70S 30S
16S RNA
21 proteins
50S
23S & 5S
RNAs
31 proteinsRat
cytoplasm
80S 40S
18S RNA
33 proteins
60S
28S, 5.8S, &5S
RNAs
49 proteins
Ribosome Composition (S = sedimentation coefficient)
7/31/2019 223 Course1
31/32
Structure of theE. coli Ribosome
The cutaway view at right shows positions of tRNA (P, E
sites) & mRNA (as orange beads, data from Joachim
Frank lab at the Wadsworth Center)
small subunit
large subunit
mRNA
location
EF-G
tRNA
7/31/2019 223 Course1
32/32
The cutaway view at right shows that the tunnel in the
yeast large ribosome subunit, through which nascentpolypeptides emerge from the ribosome, lines up with the
lumen of the ER Sec61 channel.
small
subunit large
subunit
Sec61 channel
path of
nascentprotein