Transcript Bio 251 07 TLN Genet..

Single-letter code:
M
D
L
Y
The Genetic Code
How the genetic code was deduced is quite an interesting but
horribly complicated story of prokaryotic genetics. I’ll just give
you the Cliff notes version:
Francis Crick and Sidney Brenner figured out that:
The genetic code maps ‘codons’ of 3 bases into one
amino acid.
AUA
->
Ile
GAU
->
Asp
AGA
->
Arg
mRNA
->
Amino Acid
The Genetic Code
Crick and Brenner figured
out that:
The DNA code is read
sequentially from a fixed
position in the gene
The mechanism and machinery for translating a protein
Three components:
Translation Requires:
Ґ Message in the form of
mRNA Й
Ґ A RibosomeЙ ..
Ґ Another type of RNA Й called
Transfer RNA ( tRNA)
Ґ A pool of amino acids in
cytoplasm
AA1
AA2
AA3
Free Amino Acids
mRNA, rRNA, tRNA and protein synthesis
In translation, the language of nucleic acids is translated into a new
language, that of proteins
mRNA provides the code, in linear digital form, for making a protein
tRNA provides an adaptor that links the code in a polynucleotide chain
to amino acids that make up the polypeptide chain
rRNA and ribosomes provide the decoder. Ribosomes bring together
mRNA and tRNA, and catalyze the translation of an mRNA into a
polypeptide chain. Ribosomes are the site of protein synthesis.
Ribosomes create peptide bonds between amino acids to create proteins
tRNA is the adapter.
Amino acid is matched to
the Anti-codon.
Complementary to the Codon
3’
5’
Two views of the adaptor molecule,
transfer RNA (tRNA), which guides
amino acids to the mRNA-ribosome
complex
5’
3’
The anticodon of the tRNA aligns with the codon in mRNA through
complementary base pairing
Translation occurs 5’ to 3’
Is tRNA orientation
with mRNA correct
as drawn?
Translation is performed with the help of the Ribosome.
RNA is the major component of
the Ribosome.
About 2/3 of the
Ribosome is RNA
by mass. These
RNA molecules
are called rRNA
and they play a
central role in the
translation of
mRNA into
polypeptides.
Ribosomal RNAs (rRNAs)
form complex
2o and 3o structures
that are essential for
their function
What does this rRNA
molecule look like in
three dimensions?
3-D model of 16s rRNA
molecule as it folds in the
ribosome…
…and overlaid with
its protein subunits
Behold, the large subunit
containing the 23s + 5s
rRNAs
Proteins are blue, RNAs are red
and white
Translation - Initiation
fMet
Large
subunit
E
P
A
UAC
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
Small mRNA
subunit
3’
Translation - Elongation
Polypeptide
Arg
Met
Phe
Leu
Ser
Aminoacyl tRNA
Gly
Ribosome
E
P
A
CCA
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
mRNA
3’
Translation - Elongation
Polypeptide
Met
Phe
Leu
Ser
Gly
Arg
Aminoacyl tRNA
Ribosome
E
P
A
CCA UCU
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
mRNA
3’
Translation - Elongation
Polypeptide
Met
Phe
Leu
Ser
Gly
Arg
Ribosome
E
P
A
CCA UCU
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
mRNA
3’
Translation - Elongation
Polypeptide
Met
Phe
Leu
Ala
Ser
Gly
Aminoacyl tRNA
Arg
Ribosome
E
P
A
CCA
UCU
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
mRNA
3’
Translation - Elongation
Polypeptide
Met
Phe
Leu
Ser
Gly
Arg
Ribosome
E
Ala
P
A
UCU CGA
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
mRNA
3’
Translation - Termination
Met
Phe
Leu
Ser
Gly
Polypeptide
Arg
Ala
Ribosome
Val
E
P
A
CGA
CGA
GCA...TAAAAAA
STOP
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT
mRNA
3’
Translation - Termination
Met
Phe
Leu
Ser
Gly
Polypeptide
Arg
Ala
Val
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 3’
mRNA
STOP
Translation normally
occurs on polyribosomes,
or polysomes
This allows for amplification
of the signal from DNA and
RNA, i.e.,…
One gene copy
Hundreds of mRNAs
Thousands of proteins
Translation: the movie
How many bases are required to make a genetic code to
serve 20 different amino acids?
# of 2-base combos = 42 = 16
Not enough!
# of 3-base combos = 43 = 64
Too many!
What is the solution?
How?
Evolve a code that is redundant!
Degeneracy at the third codon position
Let’s look at the Genetic Code
Transfer RNA (tRNA) and
the genetic code
Many different
base modifications
occur in tRNA
Why are these
modifications
necessary?
Transfer RNA (tRNA) and
the genetic code
Legitimate
G-U bp
Wobble bp
Inosine can pair legitimately
with three other bases, when inosine
is in the wobble position, i.e., the
first, or 5’ base of the anticodon
How many chemical changes are
required to convert guanine to
Inosine?
How many chemical changes are
required to convert adenine to
Inosine?
Is there a logic to the Genetic Code?
8 codon families where the third codon position is fully degenerate,
i.e, any of the four bases has the same meaning: (4-fold degeneracy)
AC__
Thr
GC__
Ala
CU__
Leu
UC__
Ser
GG__
Gly
GU__
Val
CC__
Pro
CG__
Arg
Thus, for these 8 amino acids, substituting one base for
another at the 3rd position is a SYNONYMOUS change
Is there a logic to the Genetic Code?
7 codon pairs where meaning is the same whichever pyrimidine occurs at
3rd position: (2-fold degeneracy)
UAU
UAC
Tyr
UGU
UGC
Cys
AGU Ser
AGC
UUU
UUC
Phe
CAU
CAC
His
GAU Asp
GAC
AAU
AAC
Asn
5 codon pairs where purines in the 3rd position are interchangeable:
(2-fold degeneracy)
AGA
AGG
Arg
AAA
AAG
Lys
GAA
GAG
Glu
CAA
CAG
Gln
UUA
UUG
Leu
Therefore, half of 3rd position substitutions will be SYNONYMOUS
and half will be NON-SYNONYMOUS
The logic of the Genetic Code
8 families with 4-fold degeneracy = 32 codons
12 families with 2-fold degeneracy = 24 codons
This accounts for 17 amino acids:
- Trp (UGG) and Met (AUG) are specified by unique codons (non-degenerate codons)
- What’s left?
-- How is Ile specified?
Isoleucine (Ile)
(This is one of today’s homework questions)
+ 3 STOP codons (UAA, UGA, UAG)
= a 64 total codons
Point mutations
Point mutations can affect protein structure and function
Type of point mutations:
- substitutions (missense and nonsense mutations)
- insertions and deletions (frameshift mutations)
Frame Shift Mutations
What happens when you get insertions or deletions of bases in
the DNA sequence?
Usually you end up with a mess.
THE BIG FAT CAT ATE THE RAT AND GOT ILL
Deletion of one base
THE IGF ATC ATA TET HER ATA NDG OTI LL
And its all pops and buzzes.
Usually frame shift mutations result in premature stop codons.
Relationship between genes and proteins
Example of a monogenic, or single-gene disorder
What is wrong with the mouse on the right?
Alkaptonuria, aka Black Urine disease, in humans
Symptoms:
- urine and ear wax turns black as ink shortly after exposure to air
-ochronosis: musculoskeletal effects, including progressive
degenerative arthritis of the large joints, esp. hip and shoulder
- signs of ochronosis:
-black deposits in the sclerae (white of eye)
-blue colored auricles (ear lobes)
What is the gene defect, or molecular genetic basis,
in alkaptonuria?
- four point mutations occur in the human HGO gene
- Blastp comparison of HGO gene from human and fungus
RVTLPDGPVRGYICELYQGHYQLPELGPIGSNGLANARDFQAPVAAFDDEEGPTE 247
+ + +
RGYI E+Y H++LP+LGPIG+NGLAN RDF P+A ++D + P
SIDVFE-ETRGYILEVYGVHFELPDLGPIGANGLANPRDFLIPIAWYEDRQVPGG 242
T196fs (frameshift)
P230S (missense)
KFNNHLFSARQDHTPFDIVAWHGNYYPYKYDLGRFNTMGSVSFDHPDPSIYTVLT 307
K+
LF+A+QD +PF++VAWHGNY PYKY+L F + SV+FDHDPSI+ TVLT
KYQGKLFAAKQDVSPFNVVAWHGNYTPYKYNLKNFMVINSVAFDHADPSIFTVLT 302
(missense) V300G
VGTAIADFVIFPPRWLVAEKTFRPPWYHRNTMSEFMGLITGNYDAKTGGGFQPAG 367
G AIADFVIFPPRW VA+KTFRPP+YHRN MSEFMGLI G+Y+AK GGF P G
PGVAIADFVIFPPRWGVADKTFRPPYYHRNCMSEFMGLIRGHYEAKQ-GGFLPGG 361
R321X (nonsense)
Are any of the sites in these proteins functionally constrained?
Molecular basis of Alkaptonuria
Mutations:
Proline230Serine and Valine300Glycine account
for most cases of the disorder
Haplotypes: each of these two mutant alleles constitutes a
haplotype, i.e., one haploid type. Progeny inherit
one haplotype from each parent.
Categorize three different types of point mutations
1
GAA
… ATA TAC GTA CAT …
… ile tyr val his …
glu
2
3
TGC
TAG
… AAA GAA TGG GTT …
… lys glu trp val …
cys
… TCG CCA TGG CCA …
… ser pro trp pro…
X (stop)
Chemical
type:
Transversion
Transversion
Transition
Informational
type:
Missense
Missense
Nonsense
Functional
type:
Deleterious
Non-synonymous
Nondegenerate site
Deleterious
Non-synonymous
Nondegenerate site
Deleterious
Non-synonymous
Nondegenerate site
Categorize three more types of point mutations
4
5
GTC
… ATA TAC GTA CAT …
… ile tyr val his …
6
TGC
A
… AAA GAA TGT GTT …
… lys glu cys val …
val
cys
… TCG CCA TGG CCA …
… ser pro trp pro …
… TCG CAC ATG GCC A …
… ser his met gly
…
Chemical
type:
Transversion
Transition
Indel (insertion)
Informational
type:
Missense
Missense
Frameshift
Functional
type:
Neutral (silent)
Synonymous
(4-fold degenerate site)
Neutral
Synonymous
(2-fold degenerate site)
Deleterious
Non-synonymous
Is a 4-fold degenerate site ever functionally constrained?
Where can you get more information about the basic concepts
embedded within the Central Dogma of Molecular Biology?
Here is a great site, full of simple, clear, and animated (!) tutorials:
http://www.dnaftb.org/dnaftb/
Chapters 15-28 in this series provides an excellent review of the first
group of lectures in Bioinformatics:
http://www.dnaftb.org/dnaftb/15/concept/
Homework #5 : due Monday, Feb. 12
Express fractions as a %
Ribosomes are large
ribonucleoprotein (RNP)
complexes
They are complex affairs,
composed of an array of
RNA + proteins