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

Nucleic Acids Protein

The Basic Blocks of Life

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)”

However, there are notable exceptions to the normal pathway of information flow!

1

2

3

Solid arrows represent "probable" information flow. Dotted arrows represent "possible" information flow.

1: DNA replication

2: Transcription

3: Translation!

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)

mRNA: AUGAGUAACGCG

Protein: MetSerAsnAla (Chain of amino acids)

Transcription

Translation

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.  Types of RNA polymerases. b.  Promoters, enhancers & silencers. c.  General transcription factors. d.  Transcription initiation complex. e.  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

Mushroom can kill!! Do not eat mushroom from the genus amanita!!

1. Transcription: Types of RNA polymerases

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)

Eukaryotic RNA Pol activities: Catalyze transcription in a 5’ to 3’ direction and synthesize RNA complementary to the template strand.

1. Transcription: Types of RNA polymerases

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-Pro-Thr-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

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

1. Transcription: Promoters, enhancers & silencers

TATA Box Upstream

Elements

Enhancers/

Silencers

-1 kb -100/-200bp -25/-30 bp +1 bp

Position- and orientation-independent

Promoter

Start of transcription:

AUG

DNA Strand

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: General Transcription Factors

1. Transcription: Transcription Initiation Complex

1.  It is like DNA replication in that a DNA strand is used to synthesize a strand of mRNA.

2.  Only one strand of DNA is copied. 3.  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.

1. Transcription: Review

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).

2. Post Transcriptional Modification

Three main forms of post-transcriptional modification 1.  5’ capping 2.  3’ polyadenylation 3.  Splicing

Def: Addition of 7-methylguanosine (m7G) residue at 5’ end pre-mRNA before the chain becomes more than 20-30 nt long.

2. Post Transcriptional Modification: 5’ Capping

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.

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: 5’ Capping

Def: The covalent linkage of a polyadenylyl moiety to the 3’ of pre-mRNA molecule to produce a poly(A) tail.

2. Post Transcriptional Modification: Polyadenylation

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’

Functions of 3’ end Polyadenylation:

1.  Protects the mRNA molecule from exonucleases 2.  Important for transcription termination 3.  For export of the mRNA from the nucleus 4.  For efficiency during translation !

2. Post Transcriptional Modification: Polyadenylation

Def: The process of cutting the pre-mRNA to remove the introns and joining together the exons.

2. Post Transcriptional Modification: Splicing

Types of splicing

1.  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.

2.  Self-splicing: Occurs for rare introns that form a ribozyme, performing the functions of the spliceosome by RNA alone.

3.  tRNA splicing: Usually occurs in tRNA. Ribonucleases cleave the RNA and ligases join the exons together. Does not require any RNA components for catalysis.

Biochemical Mechanism Spliceosomal splicing and self-splicing involves a two-step biochemical process. Both steps involve transesterification reactions that occur between RNA nucleotides!

2. Post Transcriptional Modification: Splicing

1.  First, a specific branch-point (AG) nucleotide within the intron reacts with the first nucleotide of the intron (GU), forming an intron lariat.

2.  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.!

AG GU

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.!

2. Post Transcriptional Modification: Splicing

It invalidates the old theory of one DNA sequence coding for one polypeptide (the "one-gene-one-protein" hypothesis).!

Splicing errors Mutations in the introns or exons can prevent splicing and thus may prevent protein biosynthesis.

2. Post Transcriptional Modification: Splicing

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.

1.  Definitions & preview. 2.  The genetic code. 3.  The players.

a. tranfer RNA (tRNA) b. Ribosomes

4.  Mechanism of protein synthesis

3. Translation: General Overview

Def: The process by which ribosomes use the information in mRNAs to synthesize protein.

3. Translation: Definition & Preview

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)

mRNA: AUGAGUAACGCG WHAT DOES IT MEAN?

IT IS A CODE FOR PROTEIN.

HOW DOES IT WORK?

Transcription + Post-Transcript Modification

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

3. Translation: Da Genetic Code

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

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.

3. Translation: The Main Players

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.

2.  The Ribosomes An organelle composed of rRNA and ribosomal proteins where mRNA is being translated into a protein.

3. Translation: The Main Players

Eukaryotic ribosomes are bigger than the prokaryotic ribosomes. But the eukaryotic mitochondria & chloroplast have ribosome with similar size as the prokaryotic (endosymbiotic theory).

40s

60s

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)

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).!

3. Translation: Mechanism of Protein Synthesis

Initiaton Factors (IF):

•  eIF1A, eIF3: Binding to ribosome subunits.

•  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

2. Elongation Repeated cycles of amino acid addition

3. Translation: Mechanism of Protein Synthesis

eEF1α

eEF1α

aa-tRNA-eEF1α-GTP complex

Regeneration of eEF1α-GTP

eEF2 eEF2

Elongation Factors (EF): eEF1α: Aminoacyl tRNA delivery to ribosome eEF2: Translocation

Peptidyl transferase makes a peptide bond to join the two amino acids.

eEF2 is a translocase, moves the ribosome one codon along the mRNA

3. Translation: Mechanism of Protein Synthesis

eRF

eRF

3. Termination The release of the polypeptide chain

Release Factor (RF)

eRF: Polypeptide chain release

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.

REVIEW

Post-Transcriptional Modification

Nucleus

Nucleus

Cytoplasm

The End