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DNA  Protein
Mutations
Reading
Ch 17: Gene Expression
Transcription
Transcription
• non-coding DNA
- Promoters
- Splicing: exons/introns
• RNA structures
Translation
• tRNAs
• Ribosome
• splicing
• ribozymes
Homework
Ch 48 Prequiz
mutations can affect protein
structure and function
• Mutations are changes in the genetic material of a
cell or virus
• Point mutations are chemical changes in just one
base pair of a gene
• The change of a single nucleotide in a DNA
template strand can lead to the production of an
abnormal protein
How do mutations occur?
Small changes can be devestating
R = CH2CH2-COOH
R = CH-(CH2)3
Types of Point Mutations
• Point mutations within a gene can be divided into
two general categories
– Base-pair substitutions
– Base-pair insertions or deletions
More…
Deletion
The following would be a:
5’-C AUG CCU AGG UAG G-3’
Met-Pro- Arg stop
5’-C AUG GCU AGG UAG G-3’
Met-Asp - Arg stop
a) Silent mutation
b) Missense
c) Nonsense
d) frameshift
Gene Structure
Genes have a promoter, coding region and
termination region
Genes are more than code for protein
Prokaryotic RNA polymerase binds promoters
Initiation
Terminator
Termination point
Coding Sequence
Upstream of gene
Downstream
Gene A
Gene X
Gene Q
Although no promoter shown,
it is implied that there is one
=
Gene A
Gene X
Gene Q
=
Pro
Gene X
Promoter
Gene X
•Promoter indicates direction
•Arrow indicated where transcription begins
•ALL genes have a promoter
Which strand is the template strand?
a) Top
b) Bottom
c) Neither there is no template strand in
transcription
Synthesis of an RNA Transcript
• The three stages of transcription:
– Initiation (our focus)
• melting and 1st nucleotide
– Elongation
– Termination
RNA Polymerase
•separates 2 strands and initiates transcription
•No need for primer
•Uses NTPs to start and build (polymerize) RNA
Eukaryotic RNA Polymerase II
Binding and Initiation of Transcription
• Promoters signal the initiation of RNA synthesis
Basal level
• Transcription factors mediate the binding of RNA
polymerase and the initiation of transcription
• The completed assembly of transcription factors
and RNA polymerase II bound to a promoter is
called a transcription initiation complex
• A promoter called a TATA box is crucial in forming
the initiation complex in eukaryotes
– Fewer hydrogen bonds – easier to melt
In eukaryotes
transcription
factors required
Transcription bubble does NOT go both ways
After RNA pol moves – DNA behind it renatures
Nontemplate
Elongation
No need for:
•Helicase
•topoisomerase
RNA
polymerase
3’
RNA nucleotides
3’ end
5’
5’
Newly made
RNA
Direction of
transcription
(“downstream”)
Template
Elongation of the RNA Strand
• As RNA polymerase moves along the DNA, it untwists the double
helix, 10 to 20 bases at a time
• 40 nucleotides per second in eukaryotes
• A gene can be transcribed simultaneously by several RNA
polymerases
• prokaryotes translation can occur simultaneously with
transcription due to the lack of a nuclear membrane
Termination
• Bacteria: polymerase stops transcription at end of terminator
• In eukaryotes, the polymerase continues transcription after the
pre-mRNA is cleaved from the growing RNA chain; the polymerase
eventually falls off the DNA
AUG
ppp- Translation start
UAA
-OH
UAG
UGA
Translation stop
Prokaryote gene/mRNA Structure
Transcription
Initiation
Transcription
Termination
promoter
DNA
5’ untranslated
mRNA
5’-PPP
3’ untranslated
AUG
Translation
Start
Ribosome binding site
Shine-Delgarno (only in bacteria)
UAA
Stop
3’-OH
Eukaryotes: Alteration of mRNA Ends
• Each end of a pre-mRNA molecule is modified:
– The 5 end receives a modified nucleotide 5 cap
– The 3 end gets a poly-A tail
• These modifications share several functions:
– They seem to facilitate the export of mRNA
– They protect mRNA from hydrolytic enzymes
– They help ribosomes attach to the 5 end
RNA processing in Eukaryotes
Eukaryote genes also have exons/introns
Primary Transcript: or pre-mRNA
After Transcription, before leaves nucleus
Guanosine
Cap added
G-PPP
Splicing components
• snRNA – small nuclear RNA
• snRNP – small nuclear ribonucleo-protein complex.
Each has one snRNA and about 7 different proteins.
• There are 5 different snRNPs, each with its own
distinct snRNA (U1, U2, U4, U5, or U6) and distinct
proteins.
• Spliceosome – complex of all the snRNPs that work
together to mediate splicing
Biology Themes Reminder:
Proteins do “work” / Proteins have “function”
• ie – most tasks are completed by proteins
• ex: G3P
G3P dehydrogenase
BPG
Why are proteins functional?
Biology Themes Reminder: Structure = Function
• Proteins have diverse shape
• The shape gives a unique (specific) pocket where
interaction can occur
• The active site is
where specific
electrons movement
(chemical reactions)
occur between
catalyst and reactants
Proteins can interact with DNA in a sequence
specific manner
But DNA is relatively uniform, so
not always easy
Protein
DNA nucleotides
Ribozymes
• Ribozymes are catalytic RNA molecules that
function as enzymes and can splice RNA
• The discovery of ribozymes rendered obsolete the
belief that all biological catalysts were proteins
• 3 properties of RNA enable it to function as enzyme
– It can form a three-dimensional structure because of
its ability to base pair with itself
– Some bases in RNA contain functional groups
– RNA may hydrogen-bond with other nucleic acid
molecules
RNA has many functional (reactive) groups
1 more OH (hydroxyl/alcohol group) compared to DNA
Base
Phosphate groups
H
Ribose
Deoxyribose
RNA has shape
Shape = function like protein
•
Not as many as proteins though
•
•
•
Some areas are single stranded
Some Double stranded (anti-parallel regions)
DNA is mostly double-stranded (helix) and
fewer options for shapes
1 unique aspect / advantage over protein is that
because it is RNA it has a sequence.
This allows it to bind other sequences with relative
ease versus a protein, which a whole chain for a few
interactions
All of the rest of
protein needs to
have a
complimentary
For proteins
shape
to bind here
snRNA acts like
a linker
snRNA binds mRNA
and protein
Proteins link to each
other
Only a set of proteins
needed
Rather than a different
protein for every splice
site as with restriction
enzymes
Loop
lariat
snRNAs are gene products
DNA
mRNA
Protein
NOT ALL GENES BECOME PROTEINS!
The Functional and Evolutionary
Importance of Introns
• Some genes can encode more than one kind of
polypeptide, depending on which segments are
treated as exons during RNA splicing
• Such variations are called alternative RNA splicing
• Because of alternative splicing, the number of
different proteins an organism can produce is much
greater than its number of genes
Alternative splicing
Domains
• Proteins often have a
modular architecture
consisting of discrete regions
called domains
– can have a isolated function
– Ex: DNA Pol I has a domain for
exonuclease activity and a
separate domain for
polymerase activity
• In many cases, different
exons code for the different
domains in a protein
• Exon shuffling may result in
the evolution of new proteins
Domains
• Proteins often have a
modular architecture
consisting of discrete regions
called domains
– can have a isolated function
– Ex: DNA Pol I has a domain for
exonuclease activity and a
separate domain for
polymerase activity
• In many cases, different
exons code for the different
domains in a protein
• Exon shuffling may result in
the evolution of new proteins
Translation: The Ribosome &
transfer RNAs (tRNAs)
The Ribosome
• catalyzes peptide bond formation
(joins amino acids)
• Houses mRNA & tRNAs
tRNAs determine what
amino acid is used
Carry correct amino acids
to corresponding codon
Intro
Ribosome
• Ribosomes facilitate specific coupling of tRNA anticodons with
mRNA codons in protein synthesis
• The two ribosomal subunits
rRNA gives ribosome shape
Large subunit
• 31 proteins
• 2 rRNA
23S (~3000nt’s)
5S (~300 nt’s)
Enzymatic
Small subunit
• 20 Proteins
• rRNA
16S (~1500 nt’s)
Forms bottom of A P E sites
 Where codons and
anticodons interact
Ribosomal proteins and rRNAs are encoded by genes
rRNAs are NOT translated though
Building a Polypeptide
• The three stages of translation:
– Initiation
– Elongation
– Termination
• All three stages require protein “factors” that aid in
the translation process
Initiation
1. Prokaryotes: Shine-delgarno sequence
orients small subunit just prior to AUG
1. Eukaryotes: small subunit binds to the 5’-cap
• Translation starts at 1st AUG
• AUG  Methionine is always the first amino acid
Initiation
2. Met-tRNA binds (tenuous)
3. Large subunit attaches (Takes Energy)
Shine-Delgarno
A ribosome has three binding
sites for tRNA:
–The P site holds the tRNA
that carries the growing
polypeptide chain
–The A site holds the tRNA
that carries the next amino
acid to be added to the
chain
–The E site is the exit site,
where discharged tRNAs
leave the ribosome
Elongation
1. Codon
Recognition
3. Translocation
2. Peptide bond
formation
Termination of Translation
• stop codon in A site
• The A site accepts a protein called a release factor
• causes the addition of a H2O instead of amino acid
(Hydolysis)
• This reaction releases the polypeptide, and the
translation assembly then comes apart
Ribosome
5’- C G A U G
CCC
AAA
U A A C- 3'
UAC
E
P
Met
A
Initiation
• Ribosome binds at shinedelgarno (prok) or 5’-cap (euk)
• Translation starts at 1st AUG
• AUG  Methionine is always
the first amino acid
tRNA parts
3’
Phe
• Many tRNAs
5’
• single-stranded RNA about
80 nucleotides long
• Has 3D structure -Flattened
looks like a cloverleaf
• Amino acid attachment site
• anti-codon that binds to the
mRNA codon through base
pairing
anticodon
A U G U U C U A A
mRNA
5’- C G A U G
CCC
AAA
UAC
GGG
UUU
U A A C- 3'
Release
Factor
Hydrolysis
Met
Pro
Lys
STOP
Dehyration Synthesis / Condensation Reaction
5’- C G A U G
CCC
AAA
U A A C- 3'
Release
Factor
Met
Pro
Lys
STOP
Ribosome
mRNA
5’- C G A U G
CCC
AAA
U A A C- 3'
UAC
Met
Each tRNA has a distinct
anticodon sequence that
will base-pair with a
complementary codon
on mRNA
The methionine tRNA has an anticodon of UAC
Ribosome
5’- C G A U G
CCC
UAC
GGG
Met
Pro
AAA
U A A C- 3'
Accurate translation
requires a correct match
between the tRNA
anticodon and an mRNA
codon
Which of the following
is an anticodon for Asn?
a) UAA
b) AUC
c) UUA
d) CGU
Careful! Don’t use anticodon
on the codon table
Ribosome
5’- C G A U G
CCC
AAA
UAC
Met
Technically 3’UAC5’ (antiparallel)
so sometimes read as 5’CAU3’
U A A C- 3'
Wobble: A single tRNA anticodon can be
used for multiple codons
• Flexible pairing at the third
base of a codon is called
wobble and allows some
tRNAs to bind to more than
one codon
A-C-?
U-G-I
Thr-tRNA
Wobble: A single tRNA anticodon can be
used for multiple codons
• Flexible pairing at the third
base of a codon is called
wobble and allows some
tRNAs to bind to more than
one codon
A-C-U
U-G-I
Thr-tRNA
Wobble: A single tRNA anticodon can be
used for multiple codons
• Flexible pairing at the third
base of a codon is called
wobble and allows some
tRNAs to bind to more than
one codon
A-C-C
U-G-I
Thr-tRNA
Wobble: A single tRNA anticodon can be
used for multiple codons
• Flexible pairing at the third
base of a codon is called
wobble and allows some
tRNAs to bind to more than
one codon
A-C-A
U-G-I
Thr-tRNA
tRNAs have shape
3’
5’
RNA can be single-stranded OR
double-stranded
A hairpin loop
Base pairing
Hydrogen bonding
tRNA have shape
Structure = function
some RNAs are ribozymes (RNA enzyme)
ex: ribosomal RNA (rRNA)
tRNA have shape
tRNA shape and anticodon allows it to serve as a link /
adapter between RNA and protein
• anticodon allows it to interact with mRNA through base
pairing – in a sequence specific manner
• Shape allows proteins to bind tRNA more readily
5’- C G A U G
CCC
UAC GGG
Met
Pro
AAA
U A A C- 3'
tRNAs are reused
5’- C G A U G
CCC
AAA
U A A C- 3'
UAC GGG
Met
Uncharged
tRNA
Pro
UAC
UAC
Met
aminoacyl tRNA
synthetase
Charged tRNA
= aminoacyl tRNA
Met
tRNA have shape
The tRNAs corresponding to a particular amino acid have
the same shape that is distinct from others.
example:
•All Cysteine tRNAs have same shape
•All phenylalanine tRNAs have the same shape
•The shape of Cys tRNAs is different than the shape of Phe tRNAs
Cys
Phe
Phe
Translation
• For Each codon
 1 amino acid
(ex: CCC is Pro)
HOWEVER,
• Code is redundant
1 aa has many codons
(ex: Pro can be CCU,
CCC, CCA CCG)
Similar tRNAs are
charged by the same
tRNA synthetase
Because tRNAs for the same
amino acid have the same
shape, they can all be
recognized by the same
aminoacyl-tRNA synthetase
which will attach its amino acid
ex: all theonine tRNAs will be
recognized by threonine tRNA
synthetase and have threonine
attached to them
3 Ile tRNAs – anticodons: UAA, UAG, UAU
How many shapes
are there for these
3 Ile tRNAs?
a) 1
b) 2
c) 3
d) 4
e) 5
How many aminoacyl
tRNA synthetases are
there for the 3 Ile
tRNAs?
a) 1
b) 2
c) 3
d) 4
e) 5
tRNA Structure
• Accurate translation
requires two steps:
– a correct match
between the tRNA
anticodon and an mRNA
codon
– a correct match
between a tRNA and an
amino acid, attached by
the enzyme aminoacyltRNA synthetase
Ser
anticodon
A U G U U C U A A