6 - Gene Expression - No Spoilers

Two tools to avoid procrastination 1
1. Set up a routine
Now that you know approx. how long each homework task takes,
thoughtfully schedule each task for the same day & time each week!
Why?
Make each task like a scheduled work shift that you can’t put off
Stop struggling over the choice to work or procrastinate.
(Tip: Schedule time to review ASAP after each lecture)
(Tip: Schedule breaks every 20-40 min, so each chunk of work
is bite-sized, and you won’t procrastinate or lose focus)
(Tip: Schedule heavier tasks for less-tiring days/times)
(Tip: Schedule something fun as reward for sticking to routine)
This is hard to do on your own, the first time, so…
-For a walkthrough of how to do this, see this workshop
https://youtu.be/QeMO3NtxPMw?t=2160 from Spring, 2021.
-For templates and examples from this workshop, see the Excel
version of the Weekly Hours Worksheet (posted with Lecture 1).
Biology 101 : 2
Gene expression
Learning objectives 3
(use this slide to focus your review activities, after lecture)
After review, you should be able to:
• Using a codon table, transcribe and translate a DNA sequence to protein.
• Use the codon table to predict the impact of a DNA mutation on polypeptide sequence,
protein structure, and cell phenotype
• Describe the process of transcription including the gene structure, enzymes and regulatory
components involved
• Describe the different ways in which one gene can produce more than one type of protein.
• Describe the process of translation including the mRNA structure, enzymes and regulatory
components involved
• Compare and contrast these processes in prokaryotes and eukaryotes
First… A simplified overview of gene expression (in prokaryotes) 28
Why?
-To focus on the main goal of transcription and translation (information transfer),
before diving into the detailed steps and structures (in eukaryotes), so that we don’t get
lost in the details.

How will we do it?
-We’ll follow the genetic information from a sample gene as it is transcribed from
DNA to RNA, and then translated from RNA to a protein,
-We’ll focus on Prokaryotes now, then look at Eukaryotes in detail afterwards.
Transcription (in prokaryotes) 29
A helpful overview/background:
-A chromosome contains a long piece of DNA (which is circular, in prokaryotes)
-At specific locations (or loci) on that DNA, there are individual genes that each hold
the genetic information (or ‘blueprint’) to make a specific protein/functional RNA.
-The goal of transcription is to copy the info from just one gene onto RNA.
This RNA can then be used to direct the synthesis of a polypeptide via translation.
Alternatively: Transcription synthesizes functional RNAs (e.g. tRNAs) directly,
with no translation needed.
A gene consists of 3 parts:
 the promoter sequence helps to control when and where transcription starts.
 the coding sequence is what actually gets transcribed onto RNA.
 the terminator sequence signals the end of the gene (i.e. where transcription
ends)
Step 1 = Initiation
-A number of transcription factors bind to the promotor of the gene.
 e.g. the sigma factor binds at the -10 and -35 regions.
-These transcription factors help RNA polymerase to bind properly, so it’s ready to
begin transcription at the transcription start site.
 Notice that we number the start site as +1, and so the base pairs that will get
transcribed downstream of this start site are +1, +2, +3, etc.
Step 2 = Elongation
RNA polymerase then slides along
the gene, briefly separating the two
strands of the DNA and using the template strand of DNA to form complementary
base pairs with each new RNA nucleotide, before it’s added to the growing RNA
strand.
I’ve given you the sequence for a part of a gene below, with both the -10 region and the
transcription start site (+1) included. What should the RNA sequence be?
5’-GCTATAATTGGAGTATTTGCGGAGGATCCATGATCTCGTAAGAT-
3’
3’-CGATATTAACCTCATAAACGCCTCCTAGGTACTAGAGCATTCTA-
5’
A. 5’-
GCUAUAAUUGGAGUAUUUGCGGAGGAUCCAUGAUCUCGUAAGAU-
3’
B. 3’-
CGAUAUUAACCUCAUAAACGCCUCCUAGGUACUAGAGCAUUCUA-
5’
C. 5’-GCGGAGGAUCCAUGAUCUCGUAAGAU-3’
Step 2 = Elongation
RNA polymerase then slides along
the gene, briefly separating the two
strands of the DNA and using the template strand of DNA to form complementary
base pairs with each new RNA nucleotide, before it’s added to the growing RNA
strand.
I’ve given you the sequence for a part of a gene below, with both the -10 region and the
transcription start site (+1) included. What should the RNA sequence be?
5’-GCTATAATTGGAGTATTTGCGGAGGATCCATGATCTCGTAAGAT-
3’
3’-CGATATTAACCTCATAAACGCCTCCTAGGTACTAGAGCATTCTA-
5’
A. 5’-
GCUAUAAUUGGAGUAUUUGCGGAGGAUCCAUGAUCUCGUAAGAU-
3’
B. 3’-
CGAUAUUAACCUCAUAAACGCCUCCUAGGUACUAGAGCAUUCUA-
5’
C. 5’-GCGGAGGAUCCAUGAUCUCGUAAGAU-3’
Step 3 = Termination
So how does RNA polymerase know when
it has reached the end of a gene, where it should stop
transcribing and release the completed RNA strand?
For prokaryotes:
-The termination sequence has a GC-rich region, followed
by many A nucleotides; when this sequence is transcribed:
 The GC-rich region of the resulting RNA cause it to fold
over and base-pair with itself to form a hairpin loop.
 The many A-U base-pairs that follow are less stable
(2 H-bonds instead of the 3 H-bonds of GC-pairs),
and RNA polymerase must backtrack to find a GC pair
to stabilize itself before continuing, but is blocked by the
hairpin loop, so it is stuck, and this terminates transcription.
Note: -The two strands of the DNA close up again, unchanged.
-The now-completed RNA molecules is released.
The +1 site is indicated in the DNA strand below. If RNA polymerase is moving from
right to left during transcription, which strand is the template strand?
A. Top B. Bottom C. Neither. D. Not enough information to decide
…GAATGTATGTG…
…CTTACATACAC…
Write the sequence of the first 6 RNA nucleotides:

A. 5’-UAUCAC-3’
B. 5’-CTTACA-3’
C. 5’-CUUACA-3’
D. 5’-ACATTC-3’
E. 5’-ACAUUC-3’
…GAATGTATGTG…
…CTTACATACAC…
Write the sequence of the first 6 RNA nucleotides:

A. 5’-UAUCAC-3’  We’re transcribing to the left, not to the right
B. 5’-CTTACA-3’
C. 5’-CUUACA-3’  The template below is 5’-GAATGT-3’
D. 5’-ACATTC-3’ so the RNA should be 3’-CUUACA-5’
E. 5’-ACAUUC-3’ but strands are generally written 5’ to 3’
…GAATGTATGTG…
…CTTACATACAC…
Translation (in prokaryotes) 37
What happens next, for the RNA molecule?
-If this is a gene for a functional RNA molecule (e.g. a tRNA): no translation is needed.
-If this is a gene for a protein: this mRNA must now be translated to produce a
protein
-Transcription = copying things in the same language (written with nitrogenous bases)
-Translation = converting from one language (4 bases) to another (20 amino acids)
Step 1 = Initiation
Before we begin reading the mRNA from 5’ to 3’, we need to assemble the ribosome
and the first tRNA at the right starting location. How does that happen, in
prokaryotes?
i. The small ribosomal subunit binds to Shine Dalgarno sequence, which is on the
mRNA near its 5’ end (which makes sense, since we’re going to read it from 5’3’)
ii. An initiator tRNA (carrying a modified amino acid: fMet = N-formylmethionine)
binds to the start codon, which is a few bases downstream of the Shine-Dalgarno
sequence. (So step i. makes sure that step ii. happens at the correct AUG start codon)
iii.The large ribosomal subunit joins the small subunit to complete the translation
initiation complex. We’ll discuss how these structures interact to translate RNA to
Protein later (with Eukaryotes). For now, let’s focus on the genetic information.
Steps 2 and 3 = Elongation and Termination
The Genetic Code table lets you take each 3-base mRNA codon (e.g. 5’-UCC-3’),
which is like a 3-letter word in the language of DNA/RNA, and translate it into a
specific amino acid (Ser), which is a building block for the protein we’re synthesizing.
Use this table to translate the mRNA strand (the one that we transcribed earlier), and
write down resulting amino acid sequence.
(Tips: Remember where initiation begins at a specific start codon. Also, note that
stop codons end translation, without adding any additional amino acids)
mRNA = 5’-GCGGAGGAUCCAUGAUCUCGUAAGAU-3’
A. Ala – Glu – Ala – Stop
B. Ala – Glu – Ala
C. fMet – Ile – Ser – Stop
D. fMet – Ile – Ser
E. fMet – Leu
Steps 2 and 3 = Elongation and Termination
The Genetic Code table lets you take each 3-base mRNA codon (e.g. 5’-UCC-3’),
which is like a 3-letter word in the language of DNA/RNA, and translate it into a
specific amino acid (Ser), which is a building block for the protein we’re synthesizing.
Use this table to translate the mRNA strand (the one that we transcribed earlier), and
write down resulting amino acid sequence.
(Tips: Remember where initiation begins at a specific start codon. Also, note that
stop codons end translation, without adding any additional amino acids)
mRNA = 5’-GCGGAGGAUCCAUGAUCUCGUAAGAU-3’
A. Ala – Glu – Ala – Stop
B. Ala – Glu – Ala
C. fMet – Ile – Ser – Stop
D. fMet – Ile – Ser
E. fMet – Leu
Notice the Shine-Dalgarno sequence
now underlined in the mRNA, and
the AUG start codon a bit downstream,
which is the first codon that should be
translated. In prokaryotes, this first
amino acid is the modified fMet.
Transcription and Translation (in prokaryotes) 41
Let’s review… then shake things up a bit with some PEQs and Mutations!
We’ve now followed genetic information of a gene from DNA  RNA  Protein
DNA 5’-GCTATAATTGGAGTATTTGCGGAGGATCCATGATCTCGTAAGAT-3’
3’-CGATATTAACCTCATAAACGCCTCCTAGGTACTAGAGCATTCTA-5’
RNA 5’-GCGGAGGAUCCAUGAUCUCGUAAGAU-
3’
Protein/polypeptide: fMet-Ile-Ser
What would happen to the resulting polypeptide if a mutation changed the highlighted
base pair from CG to AT?
A. Nothing (= silent mutation)
B. The polypeptide would end early (= nonsense mutation)
C. One amino acid would be different (= missense mutation)
D. After a certain point, every amino acid would
be different, and polypeptide length will also
change (= effects of a frameshift mutation)
3’
base pair from TA to CG?
3’
3’
-Sense codons code for amino acids

-Nonsense codons (i.e. Stop codons) do not
code for any amino acid.
3’
What would happen if a mutation deleted the highlighted base pair?
3’
What would happen if a mutation deleted the highlighted base pair?
5’-GCGGAGGAUCCAUGAUCUCGUAAGAU-3’
fMet-Thr-Arg-Lys-… etc.
Note: when we delete or insert a base, we still
keep reading 3 bases at a time, so this shifts our
reading frame so that each subsequent codon
become a mix of 2 original codons… which affects
Next… A detailed look at gene expression in eukaryotes (& prokaryotes) 49
Why?
-Now that you understand how DNA, RNA, and proteins are linked by the processes
of
Transcription and Translation, and seen why this matters by exploring mutations…
we can build on that core foundation as we examine Transcription and Translation in
in a bit more detail, in Eukaryotic cells.
How will we do it?
-I’ll walk you through each step of Transcription
& Translation again (with a bit more detail).
-Along the way, we’ll compare & contrast these
processes in eukaryotes & prokaryotes, to help
you understand them both better.
Transcription in Eukaryotes (compared to prokaryotes) 50
Step 1: Initiation
-In Eukaryotes, when and where transcription begins still depends on the promoter
sequence found upstream of the RNA-coding sequence, and transcription factors still
help RNA polymerase bind properly at the +1 transcription start site.
-The transcription factors, and the DNA sequences that they recognize within the
promoter sequence (e.g. a TATA box instead of the -10 and -25 regions), are different,
& the process is more complex, involving other, more-distant regulatory regions.
Prokaryotes Eukaryotes
Step 2: Elongation
-Elongation is essentially the same in all cells, with RNA polymerase building the
growing RNA strand by complementary base-pairing with the template strand of DNA
Step 3: Termination
-Eukaryotes do not use hairpin loops to terminate transcription
-They use much more complex mechanisms that you don’t need to learn for BISC 101.
Prokaryotes Eukaryotes
Transcription in Eukaryotes 53
Review with a cool video:
https://www.youtube.com/watch?v=5MfSYnItYvg (posted in video description)
(Tip: Stand up and stretch your arms and neck, while watching this 2 min video )
RNA processing (in Eukaryotes only) 54
The RNA transcript (or pre-mRNA) produced during transcription in eukaryotes is
not ready to be translated yet… it must go through 3 types of RNA processing, first:
-A 5’ cap is added, to tell the eukaryotic ribosome where to start binding.
(Remember: prokaryotes use the Shine-Dalgarno Sequence to do this)
-A Poly-A Tail is added to signal to the cell that this is a valid mRNA from the nucleus
(and not viral RNA), so it should be allowed to cross the nuclear envelope and it
should
not be destroyed by enzymes in cytoplasm (which are there toEukaryotes
destroy viral RNA)
-Non-coding regions (introns) of the RNA
transcript must be removed, & the
coding regions (exons) spliced together.
Once this RNA processing (which happens
alongside transcription) is completed, the
resulting mRNA is ready to exit the nucleus
and be translated by ribosomes in the cytoplasm.
Prokaryotes
Summary Question: Which of the follow statements is true?
A. Prokaryotic mRNA is shorter than the coding sequence of its gene.
B. Eukaryotic mRNA is shorter than the coding sequence of its gene.
C. Eukaryotic pre-mRNA is shorter than the coding sequence of its gene.
D. Eukaryotic pre-mRNA is shorter than the completed mRNA.
E. None of the above statements are true.
Eukaryotes
Prokaryotes
A cool fact: different exons can be removed (with the introns) during RNA splicing,
which means one gene can actually produce several different mRNAs, and therefore
several different proteins!
So the one gene - one enzyme hypothesis (Beadle, 1941) is false in many ways:
-Genes can encode functional RNAs
-Genes can encode polypeptides that join other parts to form functional proteins.
-One gene can make more than one polypeptide/protein (by alternative splicing)
-Not all proteins function as enzymes (e.g. hormones, transport proteins, etc.)
Translation in Eukaryotes (compared to prokaryotes) 58
Step 1 = Initiation Eukaryotes
This is quite different in Eukaryotes:
-The small ribosomal subunit binds with the initiator
tRNA first, which carries normal Methionine (Met)
instead of the modified fMet.
-This complex then starts at the 5’ cap and scans the
mRNA until it reaches the first AUG, which is the
start codon.
After that, the large ribosomal subunit binds to
complete the transcription initiation complex,
much like in prokaryotes
Prokaryotes
Step 2 = Elongation
This is generally the same in both eukaryotes and prokaryotes, but I’ll describe the
details for both of them now:
1. The next 3-base codon on the mRNA is exposed in the A-site of the ribosome, and
aminoacyl-tRNAs (i.e. tRNAs with an amino acid attached) will try to match the
codon with their anticodons.
(Meanwhile, the tRNA in the P-site of the ribosome
is bound to the previous mRNA codon, and is
holding the growing polypeptide chain)
Step 2 = Elongation
2. Once the correct amino-acyl tRNA binds to the A-site codon, the peptide chain is
added to the new amino acid, as a peptide bond is catalyzed by the ribosome.
Step 2 = Elongation
2. Once the correct amino-acyl tRNA binds to the A-site codon, the peptide chain is
added to the new amino acid, as a peptide bond is catalyzed by the ribosome.
3. Notice that it’s the newer tRNA that now holds the polypeptide chain, & the older
tRNA is empty… so we should eject the empty tRNA & shift the ribosome towards
the 3’ end of the mRNA by 3 bases, so that we can translate the next codon…
Step 2 = Elongation
4. … that’s exactly what happens. As the ribosome shifts 3-bases to the right:
 the empty tRNA slides into the E-site of the ribosome, where it is ejected.
 the newer tRNA that’s now holding the polypeptide slides into the P-site.
 the next codon of mRNA is in the A-site, waiting for the newest aminoacyl-
tRNA to arrive.
And so the cycle repeats…
…until the stop codon reaches
the A-site of the ribosome
Step 3 = Termination
Termination of translation is also similar between all cells. Here are the details:
1. There are no aminoacyl-tRNAs that match the stop codons, so a protein release
factor
binds instead.
2. This release factor releases the polypeptide from the last tRNA, and that tRNA exits
from the E-site.
3. The large and small subunits of the ribosome separate, releasing the mRNA.
What do you think happens to those ejected tRNAs that have given up their amino
acids to make this polypeptide?
A. They are digested into individual DNA nucleotides, and reused for transcription.
B. They are digested into individual RNA nucleotides, and reused for transcription.
C. They are digested into individual RNA nucleotides, and reused for translation.
D. New amino acids are randomly attached to them, and they’re reused for translation.
E. Specific amino acids are attached to them, and they’re reused for translation.
What do you think happens to those ejected tRNAs that have given up their amino
acids to make this polypeptide?
A. They are digested into individual DNA nucleotides, and reused for transcription.
B. They are digested into individual RNA nucleotides, and reused for transcription.
C. They are digested into individual RNA nucleotides, and reused for translation.
D. New amino acids are randomly attached to them, and they’re reused for translation.
E. Specific amino acids are attached to them, and they’re reused for translation.
Enzymes (aminoacyl-tRNA synthetases) attach the
correct amino acid to match that tRNA’s anticodon!
Challenge: Which of these tRNAs would you expect to be loaded with Serine (Ser)
amino acids? (Tip: remember that we translate mRNA from 5’ to 3’, and that base-
pairing is always between antiparallel strands)
A. 1 and 2
B. 2 and 3
C. 1 and 4
D. 3 and 4
E. 2 and 4
Challenge: Which of these tRNAs would you expect to be loaded with Serine (Ser)
amino acids? (Tip: remember that we translate mRNA from 5’ to 3’, and that base-
pairing is always between antiparallel strands)
A. 1 and 2
B. 2 and 3
C. 1 and 4
D. 3 and 4
E. 2 and 4
tRNA: 5’- -3’

Base-pairing mRNA: 3’-UGA-5’ 3’-AGU-5’ 3’-UCA-5’ 3’-ACU-5’
mRNA in 5’3’order: 5’-AGU-3’ 5’-UGA-3’ 5’-ACU-3’ 5’-UCA-3’
Translated amino acid: Ser none (stop) Thr Ser
Translation in Eukaryotes 68
Review with a cool video:
https://youtu.be/TfYf_rPWUdY (link posted in Youtube description of lecture video)
(Tip: Stand up and stretch your arms and neck, while watching this 3-min. video )
A quick look back: gene expression in Prokaryotes 69
Challenge: Since prokaryotc genes do not have introns that need to be spliced out of
the RNA transcript, does this mean that the entire mRNA molecule is translated?
Justify your answer.
Eukaryotes
Prokaryotes
A quick look back: gene expression in Prokaryotes 70
Challenge: Since prokaryotc genes do not have introns that need to be spliced out of
the RNA transcript, does this mean that the entire mRNA molecule is translated?
Justify your answer.
No, there are still untranslated regions (UTR) at the 5’ & 3’ ends of the mRNA:
 For the 5’ UTR: the start codon (where translation starts) is not right at the
+1 site (where transcription began)… there are some important bases in the 5’
UTR,
such as the Shine-Dalgarno sequence where the small ribosomal subunit binds.
 For the 3’ UTR: there are still many bases after the stop codon, e.g. the terminator.
Learning objectives 71
(use this slide to focus your review activities, after lecture)
After review, you should be able to:
• Using a codon table, transcribe and translate a DNA sequence to protein.
• Use the codon table to predict the impact of a DNA mutation on polypeptide sequence,
protein structure, and cell phenotype
• Describe the process of transcription including the gene structure, enzymes and regulatory
components involved
• Describe the different ways in which one gene can produce more than one type of protein.
• Describe the process of translation including the mRNA structure, enzymes and regulatory
components involved
• Compare and contrast these processes in prokaryotes and eukaryotes

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Two tools to avoid procrastination 1

lost in the details.

Write the sequence of the first 6 RNA nucleotides:

Write the sequence of the first 6 RNA nucleotides:

-Sense codons code for amino acids

tRNA: 5’- -3’

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