Download Lecture 24: the genetic code

Lecture 24: the genetic code
Key learning goals:
• Know that 90% of a cell’s energy can be expended making
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proteins
Understand how to read the genetic code table.
Be prepared to interconvert between DNA, mRNA, and
polypeptide sequences.
Understand the roles of mRNA, tRNA, and aaRS enzymes.
Be able to explain the Brenner-Crick “adaptor hypothesis.”
Be prepared to explain the secondary and tertiary structures
of tRNA.
Know how the anticodon works and what the 3’ stem does.
Understand how the genetic code is embedded in aaRS
enzymes (this is a critical point).
Understand the “wobble” hypothesis. Understand how wobble
works at the level of base-pairing.
Be able to explain why adenosine deaminase enzymes are
essential for the synthsis of wobble tRNA’s.
Understand the biochemistry of aaRS enzymes.
Understand how some aaRS’s proofread, and why.
Template-mediated flow of biological sequence information
DNA polymerase
(2%
RNA polymerase
reverse transcriptase
62%
[splicing]
ribosome
TVSXIMR
RNA-dependent
RNA polymerase
Protein synthesis is a big deal.
Proteins are the most abundant
macromolecules in the cell.
The vast majority of enzymes are proteins.
Up to 90% of a cell’s energy can be
directed toward protein synthesis.
25% of an E. coli cell’s dry mass can be
ribosomes: almost 1,000 new ribosomes
made per E. coli cell per minute!
Many important antibiotics target
protein synthesis.
David Goodsell
Transcription and translation facilitate regulation
and amplification in gene expression
One copy of the Ovalbumin gene
many Ovalbumin mRNA’s
30,000,000,000,000,000,000
molecules of ovalbumin
Definition:
The GENETIC CODE is the set of RULES
by which a linear sequence of nucleotides
specifies the linear sequence of a polypeptide.
(2%
62%
But what is the nature of the genetic code?
TVSXIMR
y one
reating
nitrous
s
are
amma), the
ons in
e code
ature),
ssible.
obins
hanges.
le out
must be
correct
) along
obvious
omma’.
‘sense’,
a-free
about half the mutants
made with base analogues
are leaky) ; (2) Streisinger lo has found that whereas
mutants
of the lysozyme of phage T4 produced by
How
I Isaid
totoyou,
when
you
lysozyme
baas-analogues
usually
leaky,
How often
often have
haveare
said
you,that
that
whenall
youhave
have
mutants
produced
by proflavin
are negative,
eliminated
the impossible,
whatever
remains, that is,
impossible,
whatever
remains,
the however
function improbable,
is completely
lacking.
must
be
the
truth.
improbable,
must
be
the
truth.
If an acridine mutant i,3 produced by, say, adding a
—Sherlock
Holmes
base, it should revert to ‘lvild-type’—Sherlock
by deleting
a bass.
Our work on revertants
of FC-0 shows that it-usually
Starlinq
point
3
,,
;$I
Overlappirq
+7
NUCLEIC
ACID *
I’
’
,-J+-~----
’
code
--’ ’ ’ ’
ETC.
1
3
Non-overlapplnq
'
Code
Fig. 1. To show the difference
between
an overlapping
code and
a non-overlappinu
code.
The short
wrticnl
lines represent
the
bases of the nucleic acid.
The czw illustrated
is for a triplet
code
Crick et al., (1961) Nature 192:1227
Reading along a DNA or RNA strand in one direction,
there are THREE possible READING FRAMES:
start
+1
+2
CATTAGGAGACTCATTAGGAGACT
CATTAGGAGACTCATTAGGAGACT
CATTAGGAGACTCATTAGGAGACT
Notes:
• In double-stranded DNA there are SIX possible reading frames: three
on the top strand, and three on the boom strand.
• A +2 change in reading frame is equivalent to moving the frame -1.
Which codons specify which amino acids?
We understand the syntax,
now we need to learn the vocabulary
Decoding the genetic code
Codon assignments
You need to understand this table, but you don’t need to memorize it.
stop.
Start
You need to understand this table, but you don’t need to memorize it.
(2%
62%
How is the genetic code implemented?
TVSXIMR
Medical Research Council Unit for the Study of
the Molecular
Structure
of Biological
Systerns,
Cavendish Laboratory,
Cambridge,
England.
ON DEGENERATETEMPLJATESAND THE ADAPTOR HYPOTHESIS
F.H.C.
Crick,
Medical Research Council Unit for the Study of
the Molecular
Structure
of Biological
Systerns,
Cavendish
A Note for
Laboratory,
Cambridge,
England.
the RNA Tie Club.
"1s there anyone so utterly
lost as he
that seeks a way where there is no way."
1955, unpublished (!)
Kai Kg'us
ibn Iskandar.
Crick: adaptor hypothesis
“…each amino acid would combine chemically,
at a special enzyme, with a small molecule
which, having a specific hydrogen-bonding
surface, would combine specifically with
the nucleic acid template.
…there would be 20 kinds of adaptor
molecule, one for each amino acid, and 20
different enzymes to join the amino acid to
their adaptors. Sydney Brenner …calls this
the “adaptor hypothesis”, since each amino
acid is fitted with an adaptor to go to the
template.
tRNA: the adaptor
(t is for transfer)
Watson-Crick
base pairing
3´ –OH
acceptor site
3´ –OH
acceptor site
anticodon
David Goodsell
tRNA secondary & tertiary structure
Watson-Crick
base pairing
RNA secondary structure: double helices similar to A-DNA
A-DNA B-DNA
tRNA
David Goodsell
RNA secondary structure: double helices similar to A-DNA
B-DNA
A-DNA
Wobble
Wobble: Alanine tRNA recognizes
GC[A/C/U] codons:
“I” is for inosine!
Wobble
How inosine promotes wobble pairing
I = inosine
hypoxanthine
inosine base:
hypoxanthine
hypoxanthine
Notice: inosine is
deaminated adenosine!
hypoxanthine
tRNA synthetases assign the codons
…there would be 20 kinds of adaptor
molecule, one for each amino acid,
and 20 different enzymes to join
the amino acid to their adaptors.
— F. Crick
David Goodsell
Do not memorize this table (unless you are very bored).
Activation of amino acid
%84
Formation of charged tRNA
EQMRSEGMH
X62%W]RXLIXEWI
44M
EHIR]PEXIHEQMRSEGMH
T]VSTLSWTLEXEWI
4MLIEX
X62%
X62%W]RXLIXEWI
%14
EQMRSEG]PX62%
Activation of amino acid
mixed anhydride
Formation of charged tRNA (I)
Formation of charged tRNA (II)
Activation of amino acid
%84
Formation of charged tRNA
EQMRSEGMH
X62%W]RXLIXEWI
44M
EHIR]PEXIHEQMRSEGMH
T]VSTLSWTLEXEWI
4MLIEX
X62%
X62%W]RXLIXEWI
%14
EQMRSEG]PX62%
Three sources of error in translation:
1. aaRS uses wrong amino acid
as substrate
+
2. aaRS selects wrong tRNA
as substrate
+
3. Ribosome selects wrong aatRNA for codon
Distribution of bases required for correct charging of tRNAs
The diameter of each
yellow circle indicates
the number of different
tRNA molecules for which
that position is an aaRS
specificity determinant.
David Goodsell
Asp-tRNA synthetase:
induced-fit of tRNA
David Goodsell
Example: why it’s hard to acheive specificity in an aaRS
Free energy (Δ∆G°´) of methylene binding
is only ~12 kJ/mol
Thus, @ equilibrium the synthetic site of Ile RS
should contain ≥1 Val for every 200 Ile
Example: why it’s hard to acheive specificity in an aaRS
But: measured misincorporation by Ile RS is
<1 Val per 3000 Ile, not >1 per 200.
Something
is increasing accuracy (reducing error) by >10-fold.
(Review) Proofreading in DNA pol/III: 3´-5´ exonuclease
Proofreading in tRNA synthetases
Proofreading by aaRS’s - example
%84
-PI
-PI67
44M
X62%
-PIEHIR]PEXI
-PI67
%14
%84
EQMRSEG]PX62%
:EP
-PI67
ATP and time are
consumed in a
“futile cycle” to
increase accuracy!
44M
:EPEHIR]PEXI
X62%
-PI67
%14
:EP
X62%
Proofreading by aaRS’s
%84
Some aaRS’s proofread
the aminoacyl-adenylate
EQMRSEGMH
X62%W]RXLIXEWI
44M
EHIR]PEXIHEQMRSEGMH
T]VSTLSWTLEXEWI
4MLIEX
X62%
X62%W]RXLIXEWI
%14
EQMRSEG]PX62%
Others proofread
the aa-tRNA
…and some don’t bother to proofread at all.
ARTICLES
sti cell
mutation
was mapped
to chromosome
8 (s
(Aars)
gene. a, Thein
amino-acid activation and tRNA aminoacylation, and an evoluacids,
no differences
viability
were observed
following
cM
^
s.e.m.).
b,
The
RP24-359N5
bacterial
artificial
chromosome
tionarily conserved editing domain for hydrolytic editing of nonaddition of alanine, histidine or methionine (P . 0.05). In contr
contains the Aars, Mrcl and Pdpr genes, of which the latter two wer
cognate amino acids (glycine or serine) from misaminoacylated
serine
dramatically increased cell death of sti/sti fibroblasts in a do
by partial digestion with NotI. Positions of primers used to genoty
Ala
(2006) Nature
443:50
(refs 10,
11). Analysis of the primary structure of AlaRS
tRNA
dependent manner relative to that observed in wild-type cultu
suggested that Ala 734 lies within the putative editing domain
(P , 0.0001). Mutant fibroblasts were marginally sensitive to glyc
(Fig. 3a). The area encompassing this residue (Fig. 3a, b (shown in
(2.59 ^ 1.22% versus 5.30 ^ 1.19% cell death in wild-type ver
affected
challenged
serine than
glycine.
Thus,
purple)) has been shown to enhance aminoacylation efficiency
mutant
cells;when
values
are meanwith
^ s.e.m.).
Thewith
specific
sensitivity
11
examined
the effect
mutation
had on
of the en
but has no role in amino-acid activation in Escherichia coli . The
serine
is consistent
withthis
defective
editing
inthe
theability
sti mutant
Al
Ala
. With
the mutant
enzyme,todea
hydrolyse
human
Ser-tRNAshow
sti Ala–Glu substitution seems to be within a loop region well outside
protein.
Also, sti/þ
fibroblasts
an increased
sensitivity
ser
Ala
was
diminished
by
a
reproducible
40–50%
(F
of
Ser-tRNA
of the active site of
the editing domain (.15 Å) but, nevertheless,
that is intermediate
to that 1of wild-type Ala
and sti/sti fibrobl
1
Jeong Woong Lee1, Kirk Beebe2, Leslie A. Nangle2, Jaeseon Jang1†, Chantal M. Longo-Guess
,
Susan
A.
Cook
,
aboveofbackgrou
contrast,
no deacylation
Ala-tRNA
within
the sequence predicted to form this domain (Fig. 3b).
(Fig.In3h).
This contrasts
with theofnormal
phenotype
sti/þ m
Muriel T. Davisson1, John P. Sundberg1, Paul Schimmel2 & Susan L. Ackerman1,3observed with either mutant or wild-type enzyme, ex
In E. coli, mutations within the AlaRS editing domain result
suggesting that environmental challenges can result in gene dos
Ala
Ala
theofpossibility
of a some
loss of
in editing specificity by the sti
Ser-tRNA
inMisfolded
increased
production
of misacylated
Gly-tRNA
effects
the sti mutation.
proteins
are associated
with several
pathological or
conditions
including neurodegeneration.
Although
(Supplementary
8).
these abnormally folded proteins result from mutations in genes encoding disease-associated
proteins
(forFig.
Defects
in
editing
ofexample,
non-cognate
amino acids from mischar
repeat-expansion diseases), more general mechanisms that lead to misfolded proteins
in
neurons
remain
largely
A
loss
of
editing
activity
should
the acids
producti
tRNAs should lead to misincorporation ofresult
these in
amino
dur
unknown. Here we demonstrate that low levels of mischarged transfer RNAs (tRNAs) can
lead
to
an
intracellular
release
of
misacylated
tRNAs.
In
vivo,
these
incorrect
prod
protein synthesis, which would in turn lead to the accumulation
accumulation of misfolded proteins in neurons. These accumulations are accompanied by upregulation of cytoplasmic
proteins.
To determine if serine-induced
protein chaperones and by induction of the unfolded protein response. We report misfolded/unfolded
that the mouse sticky mutation,
which
death
in sti/sti
cells is correlated with the accumulation of unfol
causes cerebellar Purkinje cell loss and ataxia, is a missense mutation in the editing
domain
of the alanyl-tRNA
synthetase gene that compromises the proofreading activity of this enzyme duringproteins,
aminoacylation
of tRNAs.ubiquitination
These
we analysed
of proteins in serine-trea
findings demonstrate that disruption of translational fidelity in terminally differentiated
neurons
leads
to
the
fibroblasts pretreated with mitomycin C to inhibit cell division
accumulation of misfolded proteins and cell death, and provide a novel mechanismdilution
underlying
ofneurodegeneration.
misfolded proteins (Fig. 3i). In the absence of ser
addition, polyubiquitinated proteins were greatly increased in st
The genetic code is established in the aminoacylation reactions of
particularly in the rostral
cerebellum.indicating
This degeneration
is slowly
fibroblasts,
that misfolded/unfolded
proteins accumu
aminoacyl-tRNA synthetases, where each amino acid is linked to its
progressive so that most Purkinje cells degenerate over the course of a
in these
cells.
Ubiquitinated
proteins increased in both mutant
cognate tRNA that bears the anticodon triplet of the code. The rate of
year, excluding the majority
of these
neurons
in the caudally located
misincorporation of amino acids into proteins is very low (estimated
lobule X.
wild-type cells on addition of serine to the media, indicating t
at one error in every 103–104 codons)1,2, and this high accuracy
To investigate the high
nature concentrations
of Purkinje cell loss,
we performed
of serine
cause protein misfolding even in w
results largely from the precision of aminoacylation reactions. In
immunohistochemical studies with the apoptotic markers cleaved
type cells, perhaps
by interfering
with AlaRS editing. Consistent w
addition to tRNA recognition, aminoacyl-tRNA synthetases must
caspase 3 and cleaved poly(ADP-ribose)
polymerase
(PARP). Purkinje
discriminate between amino acids in the cellular pool. Generally,
cells that were positivemisfolded
for these markers
were elevation,
observed in mutant,
protein
levels of stress-inducible, cytosolic h
amino acids with side chains that are bulkier than those of the
but not wild-type, animals
at
four
weeks
of
age
(Fig.
1h–j
and
data
shock protein 72 (HSP72) were
increased in sti/sti fibroblasts,
cognate amino acids are sterically excluded from the active sites of
not shown). Further confirmation that these cells were undergoing
on the addition
tRNA synthetases, but smaller amino acids can fit into the active site
apoptosis was obtainedalso
fromincreased
TUNEL (TdT-mediated
dUTP nickof
endserine to wild-type cells (Fig. 3
Editing-defective tRNA synthetase causes
protein misfolding and neurodegeneration
pocket and be misactivated and mischarged. These misactivated
adenylates or mischarged tRNAs are normally cleared by the editing
function of aminoacyl-tRNA synthetases, encoded by a domain that
is distinct from the domain for aminoacylation. If they are not
cleared, genetic code ambiguity is introduced (that is, a given codon
in the messenger RNA will specify incorporation of more than one
amino acid, resulting in the production of ‘statistical polypeptides’)3–6. Here we report that an editing defect in a single tRNA
synthetase in the mouse results in neurodegeneration associated with
protein characteristics consistent with heterogeneous polypeptide
production (Supplementary Fig. 1). These results provide a novel
mechanism for the generation of misfolded proteins, which are
labelling)-positive mutant Purkinje cells (Fig. 1k–m).
The sti mutation disrupts AlaRS editing
The sti mutation disrupts the Aars gene alanyl tRNA synthetase
For functional
analysis,
weon
introduced
the A734E mutation into b
A genome scan using polymorphic
microsatellite
markers
affected
the
sti
mutation
to
chromosome
8.
F2 animals initially localized
the mouse and the human enzyme, which is 92% identical (includ
Further fine mapping demonstrated that sti resided in a 1.54Ala 734)
to mouse2,932
AlaRS.
Far-ultraviolet
circular dichroism (farcentimorgan (cM) region (45
recombinants,
meioses; Fig.
Figure
3 | Mutant fibroblasts
are2aselectively sensitive to serine. a,
CD)
spectral
analysis
purified
wild-typestructures
and mutant
proteins
and Supplementary Fig.
4).domains
Expression(a)
analysis
ofof
genes
in the sti
and their
three-dimensional
(b). The amin
critical region failed toidentical
reveal any differences
between
wild-type
and
minima
and
indistinguishable
far-UV CD spec
activation
domain
(red) and
editing
mutant cerebellar RNA. However,
sequencing
of34complementary
DNA domain (purple) is modelled on
suggesting
that
theandA734E
substitution
does
not induce
ma
the synthetase
Pyrococcus
horikoshii
free-standing
AlaRS
aeolicus
AlaRS
revealed a C-to-A nucleotide
change in
the alanyl-tRNA
23
changes
local
secondary
structure
of AlaRS
, respectively.
Blue spheres
show(Supplement
the location o
domain
gene (Aars) at nucleotide
2,201
of in
thehomologue
transcript
in mutant
mice—