Regulation Of Translation

in Eukaryotes
 Points of Gene Regulation
The control of gene expression can occur at any
step in the pathway from gene to functional
protein:

1. Packing / Unpacking of DNA

2. DNA Amplification and DNA

Rearrangement

3. Transcription

4. mRNA Processing

5. mRNA Transport

6. mRNA Degradation

7. Translation

8. Protein Processing

9. Protein Degradation
 General Structure of a Eukaryotic mRNA
Some post transcriptional regulatory elements of a eukaryotic mRNA that affects
gene expression are:

• 5’ UTR mediated regulation involves:

1. 7-methyl guanine cap (Cap)
2. Upstream open reading frames (uOFRs)
3. Internal Ribosome entry sites (IRES)
4. Hairpin like structures
5. RNA –protein interactions

• 3’ UTR mediated regulation involves:

1. Poly A tail and variations in its size
2. Cytoplasmic polyadenylation elements (CPE)
3. RNA –protein interactions (multi- protein complexes)
4. Antisense RNA interactions
General Structure of a Eukaryotic mRNA
mRNA Stability
 mRNA Stability

In Bacteria, a hairpin structure in mRNA with Rho-independent termination confers stability.

The rate limiting step in mRNA degradation is the removal of 3’ Poly A tail.
 mRNA Decay Pathway in Eukaryotic Cells

A. Deadenylation- dependent pathways (Mayor pathway of mRNA decay)

B. Deadenylation- independent pathways (Minor pathways of mRNA decay)

1. Nonsense Mediated Decay (NMD)- rescues ribosomes that translate mRNAs that has nonsense codon/
unspliced introne/extended 3’UTRs
2. Nonstop Mediated Decay (NSD)- rescues ribosomes that translate mRNAs that lack a stop codon
3. No-Go Decay (NGD)- initiated when the ribosome is stalled by an RNA secondary structure or a stretch of
codons demanding charged tRNAs that are present in low abundance
4. RNAi-dependent pathway- other mRNAs are degraded by small interfering RNA

• Nonsense Mediated Decay (NMD), Nonstop Mediated Decay (NSD) and No-Go Decay (NGD) are also called
mRNA Surveillance Pathways. mRNA are checked and defective transcripts are degraded.
• Hence these pathways prevent the accumulation of aberrant transcripts and truncated proteins by ensuring
rapid decay of the mRNAs.
A. Deadenylation- Dependent mRNA Decay
 Steps In mRNA Decay

Deadenylation-Removal
of Poly A Tail

Decapping-Removal of
5’G cap

Exonuclease Decay-
Degradation by 5’ and 3’
exonuclease
Poly A Ribonuclease
AU- Rich Elements (ARE) Regulate Half Life of mRNA

ARE are defined by their ability to promote

rapid deadenylation dependent mRNA decay.
However, their sequence requirement is only
loosely conserved.
 Model for ARE Mediated Stability and Instability of mRNA
1. ARE-BP can both increase and
decrease the affinity of PABP for the
poly A tail.

3. Binding of stabilizing factors (ARE-BP), such

as HuR to ARE might block deadenylation by
increasing the affinity of the PABP for the poly
A tail.

2. Binding of destabilizing factors (ARE-BP),

such as AUF1 to ARE might promote rapid
deadenylation by reducing the affinity of the
PABP for the poly A tail.
 mRNA Decay Pathway in Eukaryotic Cells

A. Deadenylation- dependent pathways (Mayor pathway of mRNA decay)

B. Deadenylation- independent pathways (Minor pathways of mRNA decay)

1. Endoribonucleolytic Decay
2. Nonsense Mediated Decay (NMD)- rescues ribosomes that translate mRNAs that has nonsense codon/
unspliced introne/extended 3’UTRs
3. Nonstop Mediated Decay (NSD)- rescues ribosomes that translate mRNAs that lack a stop codon
4. No-Go Decay (NGD)- initiated when the ribosome is stalled by an RNA secondary structure or a stretch of
codons demanding charged tRNAs that are present in low abundance
5. RNAi-dependent pathway- other mRNAs are degraded by small interfering RNA

• Nonsense Mediated Decay (NMD), Nonstop Mediated Decay (NSD) and No-Go Decay (NGD) are also called
mRNA Surveillance Pathways. mRNA are checked and defective transcripts are degraded.
• Hence these pathways prevent the accumulation of aberrant transcripts and truncated proteins by ensuring
rapid decay of the mRNAs.
B. Deadenylation- Independent Pathways
1. Endoribonucleolytic Decay
Endoribonucleolytic Decay

3’exonuclease
3’
5’ exonuclease
5’ 3’
5’
2. Nonsense-Mediated Decay (NMD)
Eukaryotic mRNAs with Premature Stop Codons Are Targeted For
Degradation Presence of exon – junction
complexes results to
recruitment of Upf1, Upf2,
and Upf3 proteins to the
ribosome

These proteins activate a

decapping enzyme that
removes the 5’ cap and a
deadenylating enzyme
that removes the poly-A tail of
the mRNA

The uncapped and deadenylated

mRNA is then rapidly degraded
by 5’-to-3’ (Xrn1) and 3’- to- 5’
(exosome) exonucleases that are
normally unable to degrade the
(a) Translation of a normal mRNA because of the presence
of the 5’cap and poly A tail
mRNA displaces all of the exon–junction
(b) Nonsense-mediated decay.
complexes(EJC).
For Some Genes, Nonsense Mediated Decay(NMD) is Used
to Autoregulate Level of Gene Regulation

PTB protein formed

PTB induces mis-splicing in its

own mRNA leading to
removal of exon 11

PTB protein not formed as

mRNA is degraded by NMD
 3. Nonstop-Mediated Decay (NSD)
A different process called nonstop-mediated decay rescues ribosomes that translate
mRNAs that lack a stop codon in Eukaryotes
In the absence of a stop codon, the poly-A tail of the
mRNA is translated, leading to the addition of
polylysine (AAA encodes Lys) to the end of the protein

Upon reaching the 3’ end of the template, the stalled ribosome is recognized by a
complex of Dom34 and Hbs1(two proteins related to eRF1 and eRF3 ) that
stimulate ribosome dissociation and release the peptidyl-tRNA and mRNA.

After delivering Dom34 to the ribosome, Hbs1 hydrolyzes GTP and is released.

In combination with the Rli1 ATPase, Dom34 acts to

disassemble the ribosome into its two subunits and recruit an
endonuclease that cuts the mRNA upstream(5’) to the stalled
ribosome.

The resulting mRNA fragments are degraded by

5’ to 3’ exonucleases and 3’ to 5’ exonucleases.

The protein with the polylysine at the end are

unstable and is also subject to proteolysis.
 Nonstop-Mediated Decay (NSD)
An alternative pathway involving Ski7

Ribosome goes to end of mRNA and

cleaves off PABP

A complex including Ski7 protein (related to eRF3) that helps in the

release of stalled ribosome and a 3’ to 5’ exonuclease that
degrades mRNA binds any ribosome stalled at the 3’ end

Poly lysine at the end of the protein targets it to protease for

degradation
 4. No-go-Mediated Decay (NGD)
A different process called no-go-mediated decay is initiated when the ribosome is stalled by an RNA
secondary structure or a stretch of codons demanding charged tRNAs that are present in low
abundance (often referred to as rare codons)
In the absence of a stop codon, the poly-A tail of the mRNA is translated, leading
to the addition of polylysine (AAA encodes Lys) to the end of the protein

Upon reaching the secondary structure, the stalled ribosome is recognized by a

complex of Dom34 and Hbs1(two proteins related to eRF1 and eRF3 ) that
stimulate ribosome dissociation and release the peptidyl-tRNA and mRNA.

After delivering Dom34 to the ribosome, Hbs1 hydrolyzes GTP and is released.

In combination with the Rli1 ATPase, Dom34 acts to

disassemble the ribosome into its two subunits and recruit an
endonuclease that cuts the mRNA 3’ to the stalled ribosome.

The resulting mRNA fragments are degraded by

5’ to 3’ exonucleases and 3’ to 5’ exonucleases.

The protein with the polylysine at the end are

unstable and is also subject to proteolysis.
5. RNAi-Dependent Pathway
 Regulation of Gene Expression - mRNA Transport Control

• Eukaryote mRNA transport is regulated.

• Some experiments show ~1/2 of primary transcripts never leave the nucleus and are degraded.

• Mature mRNAs appear to exit through nuclear pores, but the mechanism and signals are not understood.

• The spliceosome retention model proposes that snRNPs complete with nuclear export by staying bound to spliced
introns and preventing their export, while releasing the spliced exons and allowing them to interact with nuclear
pores.

• A correct 5’ cap also appears to be required for export to the cytoplasm.

Protein Degradation - Lysosome
Protein Degradation - Proteasome
 Ubiquitin

Scientists who discovered ubiquitin mediated proteolysis---

Nobel prize in chemistry, 2004
 The Ubiquitin-Proteasome Pathway

• Ub is conjugated to proteins that are destined for degradation by an ATP-dependent

process that involves three enzymes.
• A chain of five Ub molecules attached to the protein substrate is sufficient for the
complex to be recognized by the 26S proteasome.
• In addition to ATP-dependent reactions, Ub is removed and the protein is linearized
and injected into the central core of the proteasome, where it is digested to
peptides.
• The peptides are degraded to amino acids by peptidases in the cytoplasm or used in
antigen presentation.
 Proteasome

• Human cell contains about 40,000 proteasome that degrade almost all proteins into 7-9 amino acids
fragments.
• The active part of the proteasome is within the barrel where it is shielded from the rest of the cell.
• Only way to the barrel is through the mouth which recognizes the ubiquitin, denatures the protein
with the help of ATP and takes these proteins inside the barrel once the ubiquitin is removed.
• Peptide fragments are released from the other end of the barrel.
• Only ubiquitinated proteins can enter this barrel.
 Proteolysis in Bacteria
• The protein Ubiquitin is found in numerous different tissues and organisms but not in
bacteria. It was named Ubiquitin as in Latin Ubiquitin means everywhere.

• Although bacteria and archaea do not have lysosomes, autophagic vacuoles, ubiquitin, or
26S proteasomes, they carry out very selective and highly regulated degradation of
intracellular proteins.

• These prokaryotes contain several distinct ATP-dependent proteases that mediate the
degradation of intracellular proteins. These enzymes serve functions similar to those of
the protein degradation apparatus in eukaryotic cells.

• Prokaryotic ubiquitin-like protein (Pup) is a functional analogue

of ubiquitin found in the prokaryote Mycobacterium tuberculosis. Like ubiquitin, it
serves to direct proteins for degradation.

• Pup is attached to specific lysine residues of substrate proteins by isopeptide

bonds; this is called pupylation.