Lecture 5 – RNA processing & Translation
At the end of today’s lecture, students should be able to:
Transcription (transcribe – re-arrange for a different instrument; write out)
1) Explain how eukaryotic RNA polymerases are directed to
begin and end transcription at specific sites & the general
process of transcription.
2) Describe eukaryotic mRNA processing after transcription and
how and why mRNAs decay.
Translation (from one ‘language’ to another)
3) Describe how the genetic code is used to create proteins from
DNA, via mRNA, in eukaryotes.
4) Explain why antibiotics can target bacterial RNA and protein
synthesis, but do not alter transcription and translation in our
own eukaryotic cells.
*Review for many of you (Biol 1211 & Biol 2321)
Ch13.2 & 13.4 (transcription)
Ch14.1-2, 5 (RNA molecules & processing)
Ch 15 (The Genetic Code & Translation)
Last class: Modification of RNA after transcription
Example: Pre-mRNA processing → mRNA
Summary section 14.2
Of transcription start site
^
Template strand
1. Addition of 5' cap
2. 3' cleavage and
addition of poly(A) tail
3. RNA splicing
4. Internal RNA
modifications (not
shown)
Translation Start codon
Translation Stop codon
How stable are eukaryotic mRNA molecules? Which of the
following time-frames best matches the average half-life of a
eukaryotic mRNA molecule?
a) A few seconds
b) A few hours
c) A few weeks
d) A few years
*Let’s think about what affects
RNA stability (Ch 14)
RNA is not as stable as DNA
mRNA decays!
The half-life of eukaryotic RNA: From 20–30 minutes to 20–24 hours.
After removal of the poly(A) tail by deadenylases and removal of 5’Cap by de-capping
enzymes, mRNA decay occurs in both the 5′-to-3′ (exonucleases) and 3′-to-5′ directions
(exosome).
5' cap
De-capping enzyme complex
5’–3’ exonuclease
No cap = No translation
poly(A) tail
How would mRNA stability change if you inhibit (reduce
activity of) the de-capping enzyme complex?
a) mRNA stability would increase
b) mRNA stability would decrease
c) mRNA stability would not change
RNA is not as stable as DNA
mRNA decays, and this can be regulated, often by other classes of RNA!
• Small interfering RNAs (siRNAs; from
dsRNAs) and microRNAs (miRNAs; from
miRNA genes, ss) pair to complementary
RNAs and target them for degradation or
prevent translation(~21-25 nucleotides long)
→ can be used for genetic manipulation in
the lab!
• Piwi-interacting RNAs are primarily found in
the germ cells of animals and inhibit
transposons.
(RNA Induced
Silencing Complex)
• Long noncoding RNAs (200-100,000 bp
long) – function of many unknown still, but
many involved in gene regulation.
• CRISPR RNAs are found in prokaryotes,
where they function in defense against
foreign DNA.
*We will further discuss functions of these types of RNA when we talk about biotechnology; here is a
quick preview.
CRISPR/Cas9 'genetic scissors'
Dr. Emmanuelle Charpentier and Dr. Jennifer A. Doudna
Now, “researchers can change the DNA of animals, plants and microorganisms with extremely high
precision”
Unexpected discovery – Dr. Charpentier was studying bacteria
and found a type of RNA bacteria used to protect themselves
from viruses – bacterial immune system!
Together they re-created these ‘scissors’ in vivo and engineered
them to cut any type of DNA at selected sites. Dr. Chiasson
(SMU) uses this technique in his lab!
Chapter 11
Translation
Proteins are composed of amino acids
A protein coding gene → a polypeptide
Amino acids (aa) are building blocks of proteins
-
We have 20 common aa’s
R group (side chain) confers unique chemical properties
and influences protein structure
Amino acids - joined together by peptide bonds
*What types of amino acid substitution are
most likely to change protein function?
Proteins are composed of amino acids
A protein coding gene → a polypeptide
The primary structure of proteins determines their secondary+ structure
*Gene of interest assignment –
determine if your gene
produces a protein that
interacts with other proteins
(quaternary structure)
How does a nucleotide sequence specify which
amino acids to add to a protein?
The genetic code
Codon = DNA or RNA sequence of three nucleotides forms the code to specify a particular
amnio acid/translation stop. We have a ‘triplet code’.
64 potential codons:
- 61 codons for 20 amino acids = ‘sense codons’.
- Code is ‘degenerate’
- 3 codons = stop signals
30-50 tRNAs in most organisms
- Each tRNA binds just one aa, > 1 tRNA for most aa’s
- There is not a tRNA for every amino acid encoding codon (61)…
How does a nucleotide sequence specify which
amino acids to add to a protein?
Synonymous
codons
The genetic code – triplet code = codon
-
Amino acids specified by each codon are
given by three-letter abbreviation
-
Codons are written 5′→3′, as in mRNA.
-
AUG is an initiation codon
UAA, UAG, and UGA are termination
(stop) codons
-
Code is:
- Near universal
- Degenerate (aa > 1 codon)
-
61 possible codons, but fewer tRNA types
(~51 with different anticodons in
humans). So how are these codons
translated? Wobble!
NON-synonymous
codons
How does a nucleotide sequence specify which
amino acids to add to a protein?
Codon interacts with tRNA anti-codon
The mRNA and tRNA anticodon pair in an antiparallel fashion
*Pairing between codon/anticodon can have “Wobble” → So, one tRNA can bind to more than one
codon! 3rd site pairing is weak. This allows some tRNAs to bind to multiple codons. See Genetic code to
determine what ‘wobble’ occurs.
Sixty-one codons specify 20 different amino acids.
Therefore, some amino acids are specified by more than
one codon. Which of these also is TRUE?
(select one best answer)
A. Most amino acids can be brought to the ribosome by
more than one type of tRNA.
B. A single tRNA can only ever recognize a single codon.
C. Every amino acid is encoded by at least three codons.
An mRNA has the codon 5′ UAC 3′. What tRNA
anticodon will bind to it?
a.
b.
c.
d.
e.
5′ AUG 3′
5′ GUA 3′
5′ ATC 3′
5′ CTA 3′
5′ CAU 3′
Write the codon, with correct polarity, of all mRNA codons
that will bind to the tRNA anticodon 5′ GCU 3′, considering
wobble–base pairing rules.
How does a nucleotide sequence specify which
amino acids to add to a protein?
The genetic code – triplet code = codon
Can start ‘reading’ triplet
codons in three different
places (3 frames); nonoverlapping.
https://www.khanacademy.org/science/ap-biology/gene-expression-andregulation/translation/a/the-genetic-code-discovery-and-properties
Translate each of these
using our code!
How does a nucleotide sequence specify which
amino acids to add to a protein?
The genetic code – triplet code = codon
But, only one reading frame is
correct.
Set by initiation (start) codon
(normally AUG = ____)
https://www.khanacademy.org/science/ap-biology/gene-expression-andregulation/translation/a/the-genetic-code-discovery-and-properties
How does translation occur?
Translation - eukaryotes
Making a polypeptide from mRNA – in cytosol
The translation of an mRNA
molecule takes place on a
ribosome
(complex of many proteins +
rRNA molecules).
5’ → 3’
*also has polarity
More energetically
expensive than
transcription
N = amino end
of the protein
Four main steps:
1) tRNA charging = Binding of amino acids to transfer RNAs
2) Initiation
3) Elongation
4) Termination
No longer labelled
as 5’, 3’ – think
about why!
C = carboxyl end
Translation - eukaryotes
1. Binding of amino acids to tRNAs
(same on all tRNAs)
Anticodon – binds to
codon on mRNA
(different for different
tRNAs)
tRNA charging catalyzed by aminoacyl-tRNA
synthetases – attach aa to specific tRNA
(need ATP!!)
Translation - eukaryotes
Everything needed for translation
assembles:
2. Initiation
•
•
mRNA – with 5’Cap and polyA tail
Eukaryotic Ribosome (80S large and 40S small
and subunits)
Initiation factors (not shown) & cap and
polyA binding proteins.
Initiation tRNA (Met-tRNAiMet)
GTP (energy source)
•
•
•
1)
2)
3)
4)
80S
Initiation complex recognizes the 5’ cap in mRNA
& binds (5’UTR)
Moves along until AUG (start codon)
Proteins attached to PolyA tail interact with
those binding the 5’cap
First tRNA attached to start codon in Ribosome
‘P’ site
Aminoacyl site
Peptidyl site
Exit site
40S
Translation - eukaryotes
3. Elongation (simplified)
1)New ‘charged tRNA’ binds to A site
N - end
Aminoacyl site
Peptidyl site
Exit site
1)Peptide bond forms (catalyzed by an
rRNA!) between aa’s bound to
tRNAs in P & A sites
(releases aa in P site from tRNA)
3)Translocation of ribosome down
mRNA 5’ → 3’
(tRNA in P site now moved to E and
can be released)
A site open for new tRNA, repeats.
GTP provides energy to complete
elongation
*Due to 3rd site wobble – some tRNAs pair with more than one codon
Where does the peptide bond form during translation
elongation?
a.
between amino acids in the E and A sites
b.
between the amino acid in the P site and the tRNA
c.
between amino acids in the P and A sites
d.
between the amino acids in the A site and the tRNA
e.
between the tRNA and the ribosome
Translation - eukaryotes
4. Termination (simplified)
Translation ends when a
stop codon is
encountered (not end of
mRNA, prior to 3’UTR)
N - end
no tRNA for stop codons,
so release factor moves
in instead.
C- end
N - end
Also requires GTP
(energy!)
Which of the following statements about translation is
CORRECT?
a) A special tRNA that does not have an attached amino acid binds to stop
codons to terminate translation.
b) The first three bases at the 5′ end of an mRNA are the AUG at which
translation begins.
c) The codon for methionine appears only at the beginning of the mRNA for a
protein, not in the middle or in the end.
d) In eukaryotes, the 5′ cap and the 3′ poly(A) tail are involved in translation
initiation.
e) Ribosomes move along an mRNA in the 3′ to 5′ direction.
How would translation in a eukaryotic cell be
affected by an mRNA with a missing 5’ cap?
A. The mRNA would circularize increasing the rate of translation
B. Premature termination of translation will result in a truncated
protein
C. The ribosome and translation initiation factors will not bind
and start translation
D. Initiation will preferentially occur at the second or third AUG
codon site
Translation
Differences between eukaryotic and bacterial translation can
be targeted by antibiotics
Big difference = Composition of
Ribosomes (proteins and rRNAs):
Bacteria (70S): large 50S subunit &
small 30S subunit, different rRNAs
Block peptide bond
formation + early
tRNA release
Block peptide bond
formation + other steps
Look like tRNAs - block
Eukaryotic (80S): large 60S subunit &
small 40S subunit, different rRNAs
Antibiotics – main target – bacterial
ribosomes
blocks tRNA
binding at A (30S)
bind P site,
inhibiting tRNA
binding and
formation of
initiation complex.
*Some can inhibit mitochondrial
protein synthesis - Why?
bind 30S subunit –
interfere with tRNA-rRNA
recognition, lead to
mistakes and nonfunctional proteins
https://www.biomol.com/resources/biomol-blog/how-do-antibiotics-affect-protein-synthesis
Gene regulation
During and after translation
For example, translation can be
inhibited by:
- miRNAs (often binding mRNA at
3’UTR)
- Availability of translations
machinery (e.g., tRNAs)
Can decrease protein content
Potential effects on whole-organismal phenotypes
due to differences in protein content/function