Molecular Biology Lecture 9

8/12/2019 Molecular Biology Lecture 9

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Department of Biochemistry

St. George’s University

BIOL 321 –

Translational regulation



OBJECTIVES

- Mechanisms of translational regulation

- Purposes of translational regulation- Regulation of protein folding

- Post-transcriptional regulation

- Polyadenylation

- IREs (internal ribosome entry points)

- Regulation of translation- RNA binding proteins

- 7-methyl-guanosine capping

- Post-translational regulation

- Protein stability

- Phosphorylatoin- Methylation

- Acetylation

- Glycoslyation



CRASH COURSE: Translation



Circularization of mRNA during

translation initiation:



Global

Affects most mRNAs

Due to rate limiting activities of translation initiation factors

Specific

Affects particular mRNAs

Different mechanisms

Response to

Nutrients

Developmental cues

Cell polarity/location

Mechanisms of translational regulation



A. Quality Control: Chaperones, Glycosylation

B. Degradation of misfolded proteins: Ubiquitination, ERAD

C. Proper protein function: Glycosylation, Phosphorylation,

Ubiquitination

D. Target protein to proper locations: Acylation, GPI anchors

Purposes of translational Events & Modifications



1

2

3

45

6

There are at least 6 main

mechanism involved in the

regulation of gene

expression in eukaryotes.

1) Transcriptional control

2) mRNA processing

3) mRNA transport

4) mRNA stability

5) Ribosomal selection

6) Protein stability



A. Anfinsen's dogma

All information needed for folding contained in the amino acid sequence:

- Leads to the concept of spontaneous protein folding.

B. Problems with Anfinsen's dogma (spontaneous folding)

Features of cellular environments cause misfolding & aggregation.

- Some proteins take a very long time to fold spontaneously.

- Some protein domains are prone to misfolding and aggregation.

Protein Folding



Protein folding in vivo

aggregation due to exposure of hydrophobic regions

DEAD-END PATHWAY

nascent chain

final folded structure

PRODUCTIVE PATHWAY

Folding in the cell differs from refolding of a

denatured protein in vi t ro due to:

- Linear nature of protein synthesis in v ivo .

- Exposure of hydrophobic regions duringsynthesis.

- Translation happens more slowly than folding,requiring a “delay” mechanism to allowtranslation to "catch up".

- Highly crowded cytoplasm: 300 mg/ml prot.

- Folding in vi t ro is inefficient (20 - 30%); in thecell, efficiency close to 100%.

- Conditions of stress increase misfolding andaggregation.

Problems with Anfinsen's dogma



Molecular Chaperones: Proteins that mediate correct

fate of other polypeptides but are not part of the finalstructure.

Protein Folding

History:

Molecular chaperones initially identified as heat

shock proteins, i.e. proteins upregulated by heat shockand other stresses.

Heat shock causes protein denaturation withexposure and aggregation of interactive surfaces.

Heat shock proteins inhibit aggregation by

binding to exposed surfaces during times of stress butalso during normal protein synthesis

Thus, the stress response is simply anamplification of a normal function that is used by cellsunder non-stress conditions.



Molecular chaperones

Heat Shock Proteins: Stabilize polypeptide surfaces

in an unfolded state.

Bind transiently to newly-

synthesized proteins

Bind permanently to misfolded

protein.

Have affinity for exposed

hydrophobic peptides.

Do NOT bind a specificsequence.

Regulated by ATP hydrolysis.

Undergo cycles of binding and

release

Protein Folding



Protein translation can be regulated at

several stages:

1) Post-transcription – Stem-loops, Polyadenylation, RNAi

2) At the translation step – RNA binding proteins

3) Post-translation – Protein modifications

Regulation of translationOverview:



Regulation Coded within the RNA

sequence

(Post-transcriptional regulation)



Differential mRNA polyadenylation (Ex. Mouse and Frog oocytes)

AAAAAAAAAA AAA

AAAAAAAAAA

During oogenesis:

After Oocyte

Maturation/fertlization:

Oocyte growth Cleavage

Translation begins Blocked translation

Blocked translation Translation begins

mRNAs with long poly-A tailsmRNAs have most of their poly-A

clipped off (15-90 A’s retained)

Poly-A tails are removedStored maternal mRNAs acquire

long poly-A tails (150-600 A’s)

** Specific nucleotide sequence (UUUAU: cytoplasmic polyadenylation

element) in the 3’ trailer (3’UTR) of the message marks the mRNAs to be

selectively polyadenylated at fertilization.

mRNAs encoding proteins needed for:



AAAAAAA

IRES IRES

Internal Ribosomal Entry Sites (IRES) allow for cap

independent translation.

- Viral mRNAs often contain IRES so that their mRNAs can still be

translated while disrupting translation of host cell mRNA.

- Some human mRNAs that encode proteins involved in inhibiting

apoptosis (cell death) contain IRES.

- Some human proteins can bind to mRNA IRES and alter mRNA

translation



IRE

influences onhuman

disease

Regulation of L-ferritin translation

- Only one IRE is present in ferritin mRNA

- High iron = IRPs bind iron- Low iron = IRPs are available to bind to IRE

- IRP+IRE = inhibition of ferritin translation

* Modification to ferritin IRE can affect IRP binding,

ferritin translation and IRON concentration



Hyperferritinemia cataract syndrome (HHCS)

Hereditary

- Characterized by a combination of

elevated serum ferritin in the absence

of iron overload and early onset

nuclear cataract

- Point mutations affect ability of the L-

ferritin IRE hairpin to bind IRPs, which

leads to an increase in L-ferritin mRNA

translation

- Mutations increase L-ferritin production

and increases serum ferritin levels



Regulation from

RNA binding proteins

(translational regulation)



Unfertilized egg

-mRNAs are packaged in

ribonucleoprotein (RNP)

particles

No Translation of mRNAs

-mRNAs can’t attach

to ribosomes due to RNPs

Translation of mRNA

At Fertilization

-ionic changes ( Ca++, Na+, pH)

cause release of “masking proteins”

-mRNAs are free to

attach to ribosome

Masked Messages: Ribosomes blocked by RNA binding protein



Example: PUF family of proteins-pumilo proteins in Drosophi la

AAAAAAA long (stable)

AA short

PUF

PUF complex binding site

(unstable)

Translational inhibition

- PUF proteins bind to element in the 3’ UTR of the mRNA and prevent

polyadenylation of the mRNA.

- They may also inhibit translation of the mRNA by interacting with the 5’

UTR or 7mG cap.

Masked Messages



D t i ti f th



Bcd protein forms a gradient

with highest amounts in anterior

Bcd is a transcription factor that

activates genes at different

concentrations as well aspreventing the translation of

specific mRNA

caudal mRNA

Determination of the

posterior axis of the

fruitfly:



Bicoid Protein represses caudal mRNA

translation

Bicoid

Caudal

U d RNA



Uncapped mRNAsEfficient translation requires a 7-methyl-guanosine “cap” at the 5” end of mRNA:

After Fertilization:-7-methyl added to

5’ guanosine on mRNAs

- Association with ribosome

Unfertilized oocyte:

5’ 3’

Translation

5’ 3’

7-methyl-guanosine “cap”

5’ 3’

- 5’ guanosine on oocyte mRNAs

are not methylated they can’t

attach to ribosomes

(Ex. Tobacco hornworm moth)

RNA bi di



RNA binding

proteins and

human disease-RNA-binding proteins regulate ALL

aspects of RNA biogenesis and can

be implemented in disease.

- Altered function of RBPs influences

cell cycle checkpoints, genomicstability, cancer, Neurological

disorders and Muscular atrophies.



P l i l Q li C l S l i l i



Selective proteolysis is critical for cellular regulation

- 3 steps for proteolysis in the cytoplasm:

1) Identify protein to be degraded ( with enzymes )

2) Mark it by ubiquitination

3) Deliver it to the proteasome, a protease complex that degrades it

- The Ubiquitin/Proteasome system:Ubiquitin:

- A highly-conserved 76 aa protein present in all eukaryotes

- Covalently attached to lysine side chains

- Can be a single ubiquitin or multiple branched ubiquitins

Signal for ubiquitination can be:

- Programmed via hydrophobic sequence or other motif

- Acquired by 1) phosphorylation, 2) binding to adaptor protein, or 3)protein damage due to fragmentation, oxidation or aging.

Post-translational Quality Control: Selective proteolysis.

Molecular Biology Lecture 9

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