Download HGD Gene Expression

Eukaryotic Gene Expression
The Central Dogma of Molecular
Biology and Regulation of Gene
Expression
LECTURE OUTLINE - 42 Hard Core Molecular Biology Slides!!!
The basic blocks of life
The central dogma of molecular biology
I. Transcription (DNA to pre-mRNA)
1.
Types of RNA polymerases.
2.
Promoters, enhancers & silencers.
3.
General transcription factors.
4.
Transcription initiation complex.
II. Post-Transcriptional Modification (pre-mRNA to mature mRNA)
1.
5’ capping
2.
3’ polyadenylation
3.
Splicing
III. Translation (mRNA to protein)
1.
The genetic code.
2.
The players.
3.
Mechanism of protein synthesis
Review
The Basic Blocks of Life
Nucleic Acids
Protein
The Central Dogma of Molecular Biology
“The central dogma of molecular biology deals with the detailed residue-by-residue transfer of
sequential information. It states that such information cannot be transferred from protein to either
protein or nucleic acid” (Francis Crick, Nature, 1970) .
Pathway of information flow from "DNA to RNA to protein".
“DNA makes RNA (transcription), RNA makes proteins (translation), which in turn facilitate the
previous two steps as well as the replication of DNA (replication)”
1
Solid arrows represent "probable" information flow.
Dotted arrows represent "possible" information flow.
1: DNA replication
2
3
2: Transcription
3: Translation!
However, there are notable exceptions to the normal pathway of information flow!
Overview of gene expression I
1 Replication: a double stranded
nucleic acid is duplicated to give
identical copies.
2 Transcription: a DNA segment
that constitutes a gene is read
and transcribed into a single
stranded sequence of RNA.
3 Translation: the RNA sequence
is translated into a sequence of
amino acids as the protein is
formed.
Overview of gene expression II
Gene: ATGAGTAACGCG (Non-template strand)
TACTCATTGCGC (Template strand)
Transcription
mRNA: AUGAGUAACGCG
Translation
Protein: MetSerAsnAla (Chain of amino acids)
Latest addition to the central dogma (in eukaryotic cells)
mRNA processing: Primary transcript (pre-mRNA) is processed.
It comes between transcription and translation
Eukaryotic mRNA transcript contains introns (non-coding sequence) and
exons (coding sequence)
Splicing: one or more sequences of introns are cut out while the exons
remain.
1. Transcription: General Overview
a. 
b. 
c. 
d. 
e. 
Types of RNA polymerases.
Promoters, enhancers & silencers.
General transcription factors.
Transcription initiation complex.
Review.
1. Transcription: Types of RNA polymerases
Three different polymerases transcribe different sets of genes
RNA Pol I: Located in nucleolus. Synthesize precursors of most
rRNAs.
RNA Pol II: Located in nucleoplasm. Synthesize mRNA precursors
and some small nuclear RNAs.
RNA Pol III: Located in nucleoplasm. Synthesize precursors of 5S
rRNA, tRNAs and other small nuclear RNAs.
Other types of RNA Pol in mitochondria
& chloroplast
1. Transcription: Types of RNA polymerases
Mushroom can kill!! Do not eat mushroom from the genus amanita!!
This deadly mushroom contains a toxin: α-amanitin
Upon ingestion, it binds to the RNA Pol II, effectively
causing cytolysis (destruction) of hepatocytes (liver
cells).
Diarrhea, cramps - death within a week! !
In science, the toxin is used to distinguish the three types of RNA
Polymerases
RNA Pol I: Insensitive
RNA Pol II: Extremely sensitive
RNA Pol III: Moderately sensitive
(Roeder RG et. al., 1974)
1. Transcription: Types of RNA polymerases
Eukaryotic RNA Pol activities:
Catalyze transcription in a 5’ to 3’ direction and synthesize RNA
complementary to the template strand.
DNA: 5’ ATGAGTAACGCG 3’(Non-template strand)
3’ TACTCATTGCGC 5’ (Template strand)
mRNA: AUGAGUAACGCG
Does not need precursor dNTPs, primers
Unlike pro RNA Pol, eu RNA needs additional initiation proteins to bind to
promoter.
Carboxyl Terminal Domain (CTD) of RNA Pol II:
Carboxyl end of RNA Pol II contains stretch of 7 amino acids (Tyr-Ser-ProThr-Ser-Pro-Ser); known as CTD.
Repeats essential for viability.
Focus on Pol II as it is important for the synthesis of protein coding
genes
1. Transcription: Promoters, enhancers & silencers
Promoters
Def: A DNA sequence to which RNA Pol binds prior to initiation of
transcription.
Eu promoters contain a sequence called TATA box (7 bp consensus sequence 5’ TATA[A/T]A[A/T]-3’), 25-35 bp upstream of start site of transcription.
Sequence between TATA box and the start site not important. But the spacing is
important to the start site is important.
Deletion of TATA box:
1. Loss of specificity of initiation, giving rise to many different transcripts.
2. It does not regulate the efficiency of transcription
(Grosscheld et al, 1980; Benoist et al, 1981)
3.  But for TATA box of rabbit β-globin gene - no transcription detected. Completely
destroy the promoter.
(Grosveld et al, 1982)
1. Transcription: Promoters, enhancers & silencers
TATA Binding Protein (TBP) binds to the TATA box + 1 extra DNA sequence
downstream (identity not critical)
Some promoters do not have the TATA box (Housekeeping genes)
1. 
Transcribed at low rates, initiation of transcription at different sites over a
length up to 200 bp.
2. 
The genes have GC box to compensate the lack of TATA box.
3. 
Still require TBP but with the help of other proteins for promoter to bind.
Upstream Regulatory Element (URE)
Increase the efficiency of the basal promoter
E.g. SP1 box and CCAAT box
Located 100-200 bp upstream of promoter.
Promoters may have one, both or multiple copies of these sequences.
1. Transcription: Promoters, enhancers & silencers
Enhancers
To stimulate/increase the activity of the promoters.
Mostly located thousands of bp away from the transcription start site.
Eg. SV40 enhancer: 72 bp repeats, 2 kb away of promoter of SV40 gene.
Deletion: Profound depression of in vivo transcription.
Inversion: No effect.
Change of position (Upstream/Downstream of the gene): No effect
(Chambion et al.,?)
Enhancers are orientation- and position- independent DNA elements.
Enhancer was also found within a gene, γ2b (mouse immunoglobulin).
Delete an intron, thought it had no purpose - but it decrease the amount of
transcript.
(Tonegawa et al., 1983)
1. Transcription: Promoters, enhancers & silencers
Silencers
It inhibits transcription!
How does it work?
It causes the chromatin to coil up into a condensed and inaccessible to RNA
Pol.
Initiation of transcription is inhibited thus decreasing or fully suppressing RNA
synthesis.
The neighboring genes, therefore, become inactive.
Silencers are also position- and orientation independent DNA sequences.
1. Transcription: Promoters, enhancers & silencers
Promoter
Enhancers/
DNA Strand
Silencers
-1 kb
Position- and orientationindependent
Upstream
Start of
transcription:
AUG
TATA Box
Elements
-100/-200bp
-25/-30 bp
+1 bp
1. Transcription: General Transcription Factors
Def: Proteins that regulate the activation of transcription of in the eukaryotic
nucleus.
Overview
1. 
Localize to regions of promoter & enhancer sequence elements, through
direct binding to DNA or through binding other DNA-bound protein.
2. 
Act by promoting the function of the pre-initiation complex (PIC) that recruits
& activates RNA Pol.
The most common Transcription Factors (TF):
TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, & TBP (also known as Spt15)
1. Transcription: Transcription Initiation Complex
1. Transcription: Review
1. 
2. 
3. 
It is like DNA replication in that a DNA strand is used to synthesize a strand
of mRNA.
Only one strand of DNA is copied.
A single gene may be transcribed thousands of times.
Steps involved in transcription
1. 
DNA unwinds.
2. 
RNA polymerase recognizes a specific base sequence in the DNA called a
promoter and binds to it (with helps from Initiation Factors).
3. 
The promoter identifies the start of a gene, which strand is to be copied, and
the direction that it is to be copied (5’-3’).
4. 
Complementary bases are assembled (U instead of T).
5. 
A termination code (UAA, UAG & UGA) in the DNA
indicates where transcription will stop.The mRNA produced
is called a mRNA transcript/pre-mRNA.
6.
After transcription, the DNA strands rejoin.
2. Post Transcriptional Modification
Def: A process in which pre-mRNA transcript is converted into
mature mRNA.
For prokaryotic mRNA it can be translated before it has finished being
transcribed (where?).
In eukaryotes, mRNA is synthesized as longer precursors.
The population of different pre-mRNA is known as heterogeneous nuclear
RNA (hnRNA).
Three main forms of post-transcriptional modification
1.  5’ capping
2.  3’ polyadenylation
3.  Splicing
2. Post Transcriptional Modification: 5’ Capping
Def: Addition of 7-methylguanosine (m7G) residue at 5’ end premRNA before the chain becomes more than 20-30 nt long.
Guanosine nucleotide connected to the mRNA
via an unsual 5’ to 5’ triphosphate linkage.
By guanyltransferase enzyme.
The guanosine will be methylated next.
Methylation of the 2’ hydroxy-groups of the first
3 ribose sugars of the 5’ end of the mRNA.
The 5’ cap of the mRNA looks like 3’ end of an
RNA.
2. Post Transcriptional Modification: 5’ Capping
Functions of the 5’ Cap
1. Regulation of nuclear export
Nuclear export of RNA is regulated by the Cap Binding Complex (CBC) which
binds exclusively to capped RNA. The CBC is then recognized by the nuclear
pore complex and exported.
2. Prevention of degradation by exonucleases.
Degradation of the mRNA by 5' exonucleases is prevented by functionally looking
like a 3' end. This increases the half life of the mRNA, essential in eukaryotes as
the export process takes significant time.
3. Promotion of translation
The 5’ cap is required for binding of ribosomes and translation initiation factors
4. Promotion of 5' proximal intron excision.
The 5' cap appears to loop round and interact with the spliceosome in the splicing
process, promoting intron excision.!
2. Post Transcriptional Modification: Polyadenylation
Def: The covalent linkage of a polyadenylyl moiety to the 3’ of premRNA molecule to produce a poly(A) tail.
Process:
1.  It occurs during and immediately after transcription in the nucleus.
2.  The mRNA chain is first cleaved by endonuclease complex
associated with RNA polymerase.
The cleavage site has base sequence AAUAAA near it.
3.  Finally, 50 to 250 adenosine residues are added to the free 3' end
at the cleavage site. This reaction is catalyzed by polyadenylate
polymerase.
5’- AAUAA
35 nts
AAAAAAA(50-250 bp)AAAAA-3’
2. Post Transcriptional Modification: Polyadenylation
Functions of 3’ end Polyadenylation:
1. 
2. 
3. 
4. 
Protects the mRNA molecule from exonucleases
Important for transcription termination
For export of the mRNA from the nucleus
For efficiency during translation !
2. Post Transcriptional Modification: Splicing
Def: The process of cutting the pre-mRNA to remove the introns and
joining together the exons.
Types of splicing
1. 
2. 
3. 
Spliceosomal: Catalyzed by the spliceosome
(large RNA-protein complex composed of five
small nuclear ribonucleoproteins snRNPs).
Major: 3’ & 5’ splice sites are AG and GU,
respectively
Minor: 3' and 5' splice sites are AU and AC,
respectively.
Self-splicing: Occurs for rare introns that
form a ribozyme, performing the functions of
the spliceosome by RNA alone.
tRNA splicing: Usually occurs in tRNA.
Ribonucleases cleave the RNA and ligases
join the exons together. Does not require any
RNA components for catalysis.
2. Post Transcriptional Modification: Splicing
Biochemical Mechanism
Spliceosomal splicing and self-splicing involves a two-step biochemical process.
Both steps involve transesterification reactions that occur between RNA nucleotides
!
1. 
2. 
First, a specific branch-point (AG)
nucleotide within the intron reacts
with the first nucleotide of the intron
(GU), forming an intron lariat.
GU
AG
Second, the last nucleotide of the
first exon reacts with the first
nucleotide of the second exon,
joining the exons and releasing the
intron lariat.!
!
Transesterification: a process of exchanging the alkoxy group of an ester compound by another alcohol.
2. Post Transcriptional Modification: Splicing
Alternative splicing: is a process that occurs in which the splicing
process of a pre-mRNA transcribed from one gene can lead to different
mature mRNA molecules and therefore to different protein.
!
It invalidates the old theory of one DNA sequence coding for one polypeptide
(the "one-gene-one-protein" hypothesis).!
2. Post Transcriptional Modification: Splicing
Splicing errors
Mutations in the introns or exons can prevent splicing and thus may
prevent protein biosynthesis.
Common errors:
1.  Mutation of a splice site resulting in loss of function of that site. Results
in exposure of a premature stop codon, loss of an exon, or inclusion of
an intron.
2.  Mutation of a splice site reducing specificity. May result in variation in
the splice location, causing insertion or deletion of amino acids, or
most likely, a loss of the reading frame.
3.  Transposition of a splice site, resulting in inclusion or exclusion of more
DNA than expected. Results in longer or shorter exons.
3. Translation: General Overview
1. 
2. 
3. 
4. 
Definitions & preview.
The genetic code.
The players.
a. tranfer RNA (tRNA)
b. Ribosomes
Mechanism of protein synthesis
3. Translation: Definition & Preview
Def: The process by which ribosomes use the information in mRNAs
to synthesize protein.
Prokaryotes have no nucleus, so mRNA can be translated while it is still being transcribed. The
translation is said to be polyribosomal (polysome) when there is more than one active
ribosome.
!
3. Translation: The Code
DNA: ATGAGTAACGCG (Non-template strand)
TACTCATTGCGC (Template strand)
Transcription + Post-Transcript Modification
mRNA: AUGAGUAACGCG
WHAT DOES IT MEAN?
IT IS A CODE FOR PROTEIN.
HOW DOES IT WORK?
3. Translation: Da Genetic Code
To break the genetic code we need to separate the mRNA sequence in a
number of 3 nucleotides: CODON
Code in the form of Codon
Each of those triplets codes
(codon) for a specific amino acid!
3. Translation: Da Genetic Code
1 codon: 1 amino acid (aa)
Notice stop & start codons
64 codons: 20 aa
The codes are redundant!
18/20 aa have more than 1 codon.
Only Met and Trp have single codon.
Why redundant?
To minimize the effect of mutation.
Transition: purine (AG)-purine
Transversion: pyrimidine (CU)-purine
Open Reading Frame (ORF): mRNA sequence that starts with AUG and ends with the stop
codons.
Overlapping gene: Region in mRNA where there are overlapping ORF region (e.g. phages)
The code is very nearly universal: Support hypothesis that all life has evolved from a single
common origin
The code differs slightly in mitochondria & other unicellular organism (1980)
e.g. (mitochondria): AGA/AGG (Arg)=Stop, AUA (Ile)=Met, UGA (Stop)=Trp
3. Translation: The Main Players
1. 
Transfer RNA (tRNA)
A small RNA chain (74-93 nucleotides) that transfers a specific amino
acid to a growing polypeptide chain at the ribosomal site of protein
synthesis during translation.
It has a site for amino acid attachment and a three-base region called the
anticodon that recognizes the corresponding codon region on mRNA via
complementary base pairing.
tRNAs are joined to amino acids to become aminoacyl-tRNAs (charged
tRNAs) in a reaction called aminoacylation.
Aminoacyl-tRNA synthetases carry up the joining reaction between the
tRNA and amino acids.
3. Translation: The Main Players
2. 
The Ribosomes
An organelle composed of rRNA and ribosomal proteins where mRNA is
being translated into a protein.
Consist of 2 subunits: small (40s) & large (60s)
Large ribosome has E site (Exit site), P site (Peptidyl-tRNA binding site) & A site
(Aminoacyl-tRNA binding site)
60s
40s
Eukaryotic ribosomes are bigger than the prokaryotic ribosomes. But the eukaryotic
mitochondria & chloroplast have ribosome with similar size as the prokaryotic
(endosymbiotic theory).
3. Translation: Mechanism of Protein Synthesis
There are 3 stages of protein synthesis
1.  Initiation: the assembly of a ribosome on an mRNA
2.  Elongation: repeated cycles of amino acid delivery, peptide
bond formation and movement along the mRNA.
3.  Termination: the release of the polypeptide chain.
3. Translation: Mechanism of Protein Synthesis
1. 
Initiation
Small subunit of ribosome binds to 5' end of mRNA with the help of
initiation factors (eIF).
!
Initiaton Factors (IF):
• 
eIF1A, eIF3:
subunits.
Binding
to
ribosome
• 
eIF4A, eIF4B, eIF4F: Binding to mRNA.
• 
eIF2: Initiator tRNA delivery.
• 
eIF5: Displacement of other factors.
tRNA gets into the P-site of the large ribosome.
Scanning hypothesis: 40s ribosome with tRNA
attaches to 5’ end of mRNA and scans until it
finds the start codon.
Correct sequence context: 5’-CCRCCAUG-3’
R - purine
3. Translation: Mechanism of Protein Synthesis
2. Elongation
Repeated cycles of amino
acid addition
Elongation Factors (EF):
eEF1α: Aminoacyl tRNA delivery
to ribosome
eEF2: Translocation
aa-tRNA-eEF1α-GTP
complex
eEF1α
Regeneration of
eEF1α-GTP
eEF1α
Peptidyl transferase makes a
peptide bond to join the two
amino acids.
eEF2 is a translocase, moves the
ribosome one codon along the
mRNA
eEF2
eEF2
3. Translation: Mechanism of Protein Synthesis
3. Termination
The release of the polypeptide chain
Release Factor (RF)
eRF: Polypeptide chain release
eRF
Release factor recognize the stop codon, make peptidyl
transferase join water molecule, thus releasing it.
Ribosome release factor helps to disassociate the
ribosome subunits from the mRNA.
eRF
REVIEW
Nucleus
Post-Transcriptional
Modification
Nucleus
Cytoplasm
The End