1.7 Regulation of gene expression pt 2

mRNA stability

refers to the lifespan of a mRNA molecule, which is tightly regulated

Significance of mRNA stability

Gene expression regulation
Cellular adaptation
Disease risk

Significance of mRNA stability: Gene expression regulation

The lifespan of an mRNA directly influences how much protein is produced from a given gene.

Significance of mRNA stability: Cellular adaptation

Cells can rapidly adjust protein levels by altering mRNA stability in response to changing environmental conditions or stress

Significance of mRNA stability: disease risk

The rate at which mRNA degrades is linked to disease risk

Deadenylation-dependent mRNA decay

is a mechanism that regulates mRNA stability by shortening the poly(A) tail, leading to degradation of the mRNA molecule.

Which enzyme in involved in deadenylation-dependent mRNA decay

DAN (deadenylation nuclease)

DAN (deadenylation nuclease)

associates with mRNA 5’methyl cap and degrades mRNA in the 3’ to 5’ direction.

Why is there direct competition between mRNA translation and DAN-dependent decay

both mRNA degradation by DAN and the initiation of mRNA translation use the mRNA 5’cap.

microRNA (miRNA)

short (~22 nucleotides), single-stranded RNA molecules that negatively regulate gene expression by binding to target mRNA.

How do miRNAs find target mRNA transcripts

have perfect (or near perfect) complementary base-pairing

When miRNA bind to specific sequences in a target mRNA, this

promotes cleavage of target mRNA and inhibits protein translation

where do miRNAs come from

encoded in our genome and then transcribed BUT NOT TRANSLATED

Following transcription, the primary miRNA, forms a

hairpin loop structure (= double-stranded region). This structure is then processed by the Drosha and Dicer enzymes to produce mature miRNAs.

Primary miRNA transcripts are processed into ~22- nucleotide mature miRNAs by the

Drosha and Dicer complexes

Mature miRNA associates with

Argonaute protein within a protein complex called RISC (RNA Induced Silencing Complex).

Function of RISC

guides miRNA to target mRNA.

COmplementary binding of miRNA causes

gene silencing through translational repression or mRNA degradation.

Protein synthesis thus involves interactions between 3 types of RNA molecules

ribosomes
tRNAs
mRNA template

Ribosomes are found

free in the cytosol, or associated with the endoplasmic reticulum (ER).

mRNA is associated with which ribosomal subunit

the smaller subunit during translation initiation.

Each ribosome subunit consists of

one or more ribosomal RNA (rRNA) molecule, and several ribosomal proteins (RPs)

A site of ribosome

is the location where aminoacyl-tRNA binds during protein synthesis.

P site of the ribosome

is the site where the peptidyl-tRNA is located during protein synthesis, facilitating the transfer of the growing peptide chain.

E site of the ribosome

is the exit site for the deacylated tRNA after it has transferred the peptide to the growing chain.

What molecule sets the reading frame for protein translation

is the start codon, typically AUG, which signifies the beginning of translation.

How many possible codons are there

64 (4×4×4)

61 of the 64 triplets code for

20 amino acids

3 of 64 codons

are stop codons and do not represent amino acids and cause termination.

degeneracy of the code.

Most amino acids are represented by more than one codon

anticodon site of tRNA

is the region on tRNA that pairs with the corresponding codon on mRNA during translation, ensuring the correct amino acid is added to the growing polypeptide chain.
base pairs with mRNA

amino acid attachment site of tRNA

3’ region on tRNA that attaches to a specific amino acid before it is brought to the ribosome during translation.

Since there are 64 possible codons are there 64 tRNA types

no the anticodon of one tRNA can bind with several different codons, due to wobble in the 3rd position

2 ways translation accuracy is maintained

enzymatic selection
Proofreading and editing

enzymatic selection of translation

Specific aminoacyl-tRNA synthetases (aa-tRNA synthetases) recognize and link a particular amino acid to its corresponding tRNA.

Proofreading & Editing during translation

The enzyme cleaves incorrectly paired amino acids before or after transfer to the tRNA.

Initiation of translation

requires the ribosome to bind to the mRNA, which forms an initiation complex containing the 1st aa-tRNA

The initiation of translation is regulated by the

Kozak sequence presence
Eukaryotic initiation factors

Kozak sequence

usually first AUG codon from 5’ cap site on mRNA —> optimal start site for initiation of translation

Translation initiation can be blocked by

translation repressor proteins
miRNA

translation repressor proteins

block the kozak sequence to stop initiation of translation

eukaryotic initiation factor 4 (eIf-4)

associate with mRNA transcript at the 5’ cap and 3’ polyA tail.
—> makes sure only processed mRNA is translation

Once eIF-4 associated with the mRNA

the mRNA is then correctly positioned in the small ribosomal subunit (= control step

eukaryotic initiation factor 2 (eIF-2)

bind the initiator tRNA (Met-tRNA) to the small ribosomal subunit and promotes the start of translation.

Met-tRNA only can bind

without the large ribosomal subunit attached.

Once eIf-2 brings Met-tRNA to the small subunit, then what

met-tRNA scans to find kozak (AUG) —> only then can eIF2,4 dissociate and large subunit binds

eIF-2 activity is controlled by

association with guanine nucleotide exchange factor (eIF-2B) AND phosphorylation

COnversion between active (GTP bound) eIF2 and inactive (GDP)

through the association with eIF-2B (guanine nucleotide exchange factor)

If eIF2 is phosphorylated

eIF-2B cannot function in the GTP exchange, and eIF2 remains inactive. Protein synthesis is slowed.

Elongation phase of translation

the polypeptide chain is extended by the sequential addition of amino acids

Regulation of translational elongation is mediated by

Elongation factors EF-1 and EF-2

Elongation factors EF-1 and EF-2

control the accuracy and forward drive (speed) of translation

EF-1

transports aa-tRNAs into the A site

EF-2

mediates ribosome translocation after peptide bond formation

Both EF-1 and EF-2 use

hydrolysis of GTP to GDP to:
• displace any incorrectly based-paired tRNA
• eject the spent tRNA
•reset the ribosome

termination of translation

stopping the addition of amino acids. The completed protein is released, and the ribosome dissociates from the mRNA.

Post translational control

Protein folding
Structural changes
protein processing and cleavage
post-transl modifications

alpha helix

slinky-like formations

beta sheet

zigzag patterns which resemble folds of a paper fan.

Why is protein folding such an important thing in post-transl control of gene expression

A protein’s function depends on its shape

Misfolded proteins cause several known diseases

Sickle cell anemia
Cystic fibrosis

Structural changes that mediate post-transl control

Formation of disulfide bridge which links sulphur residues between 2 cysteine amino acids and create structure.

Example of protein processing and cleavage

pre-proinsulin —> insulin

pre-proinsulin —> insulin

Insulin is synthesized as precursor protein which contains the A- and B-chain of insulin joined in a continuous single chain with an intervening C-chain. C chain is removed

post translational modifications (PMT)

Attaching biochemical functional groups to proteins (methyl, phosphate, hydroxyl, ubiquitin, etc)

Protein phosphorylation

key regulation of proteins since it is reversible change mediated by kinases and phosphatases

Ubiquitination

marks proteins for degradation via the proteasome.

1st enzymatic step of ubiquitination

Activation by ubiquitin-E1

2nd enzymatic step of ubiquitination

Conjugation by ubiquitin-E2

3rd enzymatic step of ubiquitination

Ligation by ubiquitin-E3

2 modes of protein transport

cotranslational
post-translational

Post-translational transport

Translation of mRNA occurs on free ribosomes in the cytosol then is transported to its functional cellular location

Co-translational transport

Ribosomes with mRNA attached are targeted to the endoplasmic reticulum (ER).
• Translation occurs in association with the ER.

How are ribosomes targeted to the Rough ER

signal sequence at the N-terminal of the translated protein

signal sequence

a short stretch of amino acids that interact with a complex called the signal-recognition particle (SRP).

Once signal sequence of protein (still attached to ribosome) is bound to SRP complex then what

SRP bind to receptor on rough ER, protein is synthesized INTO the rough ER, then signal is removed

Once cotranslation occurs in the rough ER, where do proteins go next

They are packaged into vesicles and sent to the Golgi apparatus for further processing.

Goli

major site for the protein modification and packaging. e.g. glycosylation (addition of oligosaccharides to membrane proteins).

Which proteins would go from golgi to secretory vesicles

Proteins destined for secretion or membrane insertion, such as peptide hormones and neurotransmitters

Which proteins would go from golgi to plasma membrane

Proteins that are incorporated into the plasma membrane, such as receptors and transporters, ion channels, cell junction proteins

Which proteins would go from golgi to lysosome

Hydrolases and integral lysosomal membrane proteins

2 types of secretion pathways (after golgi) :

Constitutive and regulated secretion

constitutive secretion

Proteins are secreted from a cell continuously, regardless of external factors or signals.

regulated secretion

Proteins are secreted from a cell when a specific signal is detected by the cell. EXAMPLE: insulin secretion

How arre proteins transported from organelle to organelle

coated vesicles

Different coats of vesicle help determine

the destination of the protein cargo during transport between organelles.

Examples of vesicle coats

Clathrin
COPI and COPII

Exocytosis of secretory vesicles

is the process by which secretory vesicles fuse with the target membrane to release their contents

The role of SNARE proteins in Exocytosis

is to mediate the fusion of secretory vesicles with the target membrane, facilitating the release of neurotransmitters or hormones.

SNAREs are transmembrane proteins that exist as

transmembrane complementary sets (vesicle SNARE—> target membrane SNARE)

v-SNARE

is a type of SNARE protein located on the vesicle membrane that forms a complex with t-SNAREs on the target membrane to promote vesicle fusion.

t-SNARE

is a type of SNARE protein located on the target membrane that pairs with v-SNAREs on vesicles to facilitate membrane fusion.

Each complimentary SNARE set is associated with

a particular organelle involved in the secretory pathway.

Botulinum toxin (BoTOX) produced by Clostridium botulinum degrades

SNARE proteins involved with docking of synaptic vesicles at the axon terminals —> no ACh release —> paralysis

Exosomes

are small extracellular vesicles that transport proteins, lipids, and RNA between cells, via membrane vesicle trafficking