Unit 7: Protein Synthesis, Processing And Regulation Flashcards
1
Q
Basics
A
- Proteins carry out functions determined by information encoded in genomic DNA
- Protein synthesis is final stage of gene expression
- mRNA translation is 1st step in the constitution of functional protein. Necessary folding + processing
- Gene expression also regulated at translational level. Mechanisms control *activity of proteins + *quantity (differential degradation)
2
Q
How are proteins synthesised from mRNA?
7.1
A
- Proteins synthesised from an *mRNA template
- mRNA read in 5’ to 3’
- polypeptide chains synthesised from amino to carboxyl terminus
- Translation takes place in ribosomes (*rRNA + proteins)
- *tRNAs are adapters between mRNA and aa incorporated into protein
3
Q
*What is the structure of Transfer RNA?
A
- 70-80 nt length
- cloverleaf shape, due to complementarity of bases between regions of molecules
- L-shaped folding
- CCA sequence at 3’ end. Aa covalently attached to ribose of terminal adenosine
- Anticodon loop: Opposite 3’ end, binds to appropriate codon by 3-base complementarity
4
Q
- What are tRNA synthetases?
7. 1
A
Selective enzymes that attach appropriate aa onto its corresponding tRNA
Mechanism summarised into following reactions:
- Amino Acid + ATP => Aminoacyl-AMP + PPi
- Aminoacyl-AMP + tRNA => Aminoacyl-tRNA + AMP
5
Q
What is the genetic code?
7.1
A
- 64 possible codons, 61 encode aa, 3 are stop codons
- Mose aa encoded by more than 1 codon, there is more than 1 tRNA for the same aa
- Some tRNAs recognise more than 1 codon: nonstandard base pairing (wobble)
6
Q
- What is nonstandard codon-anticodon base pairing?
7. 1
A
Allows G to pair with U, and inosine (I) to pair with U, C or A
(Guanosine is modified to inosine in anticodons of some tRNAs)
7
Q
- Ribosomes
7. 1
A
- *Prokaryotic 70S ribosome => 23S & 5SrRNAs (34 proteins) + 16S rRNA (21 proteins)
- *Eukaryotic 80S ribosome => 28S, 5.8S & 5S rRNAs (~46 proteins) + 18S rRNA (33 proteins)
8
Q
What is the role of rRNA in formation of peptide bonds?
7.1
A
- believed to catalyse the formation
- Necessary for in vitro assembly of functional ribosomes.
- Lack of ribosomal proteins causes a decrease but not loss of functionality
- First high res. Structural analysis (2000) shows ribosomal proteins were markedly absent from peptide bond formation site
9
Q
- What is the organisation of mRNAs + initiation of translation?
7. 1
A
- mRNAs have non coding *untranslated regions (UTRs) at the ends
- Most eukaryotic mRNAs are *mono-cistronic, encoding a single protein
- Prokaryotic mRNAs are often *poly-cistronic, encoding multiple proteins, each of which is translated from an independent start site
- *AUG codon: Start of translation with aa *methionine (in most bacteria N-formylmethionine)
10
Q
- How are initiation codons identified in prokaryotic + eukaryotic cells?
7. 1
A
- *Bacterial mRNAs: initiation codons preceded by a *Shine-Dalgarno sequence, which aligns mRNA on the ribosome. Initiated translation at 5’ end of mRNA + internal initiation sites of polycistronic mRNAs
- *Eukaryotic mRNAs: recognised by 7-methylguanosine cap at 5’ end. Ribosomes then scan downstream this cap until initiation codon encountered
11
Q
Translation process
7.1
A
- Ribosome binds to mRNA at start codon
- Polypeptide chain elongates by successively adding aa
- When stop codon encountered, polypeptide released + ribosome dissociates
12
Q
Steps of initiation of translation (bacteria)
7.1
A
- 30S ribosomal subunit binds IF1 + IF3.
- MRNA + initiator N-formylmethionyl (fMet) tRNA + IF2 (bound to GTP) join complex.
- TRNA binds to start codon. IF1 + IF3 released.
- 50S subunit associated with complex, inducing GTP hydrolysis (bound to IF2) + release of this factor
An initiation complex is formed prepared to catalyse the formation of a peptide bond during elongation.
13
Q
Steps of initiation of translation (eukaryotes)
7.1
A
- ElF1, elF1A + elF3 bind to 40S subunit.
- ElF2 bound to GTP, binds to initiator methionyl tRNA.
- Pre-initiation complex formed by associating all + elF5.
- Cap at 5’ end of mRNA recognised by elF-4E, which forms complex with elF-4A + elF-4G. elF-4A also binds to elF-4B. elF-4G also binds to poly-A binding protein (PABP).
- Factors direct mRNA to 40S subunit through interactions between elF-4G + elF-3.
- 40S subunit bound to methionyl tRNA + elF checks mRNA until AUG start codon identified. Displacement requires energy.
- When AUG recognised, elF-5 causes GTP (bound to elF-2) hydrolysis + elFs are released. elF5B (initially bound to GTP) facilitates binding of 60S subunit.
14
Q
Steps of elongation
7.1
A
- Initiator methionyl tRNA binds to P site.
- Elongation factor (EF-Tu in prokaryotes, eEF1α in eukaryotes) complexed to GTP brings aminoacyl tRNA to ribosome.
- Next aminoacyl tRNA binds to A site by pairing with 2nd codon of mRNA. Hydrolysis of GTP + release of EF.
- Peptide bond is formed, catalysed by large ribosomal subunit. Initiator TRNA (uncharged) now at P site.
- Ribosome moves 3 nucleotides along mRNA, positioning next codon in A site. Next step translocates peptidyl tRNA from A to P, and uncharged tRNA from P to E. (translocation requires EF-G in prokaryotes + eEF2 in eukaryotes, it is coupled to GTP hydrolysis).
- When next aminoacyl tRNA binds to site A, free tRNA from site E released.
15
Q
Regeneration of eEF1/GTPα (eukaryotes) or EF-Tu/GTP (prokaryotes)
7.1
A
- As elongation continues, eEF1α (or EF-Tu) released from ribosome bound to GDP must be reconverted to GTP.
- eEF1βγ (EF-Ts in prokaryotes) elongation factor required for this. It binds to the eEF1α/GDP (or EF-Tu/GDP) promoting substitution of GDP for GTP.
- New eEF1α/GTP (or EF-Tu/GTP) prepared to direct a new aminoacyl tRNA to A site of ribosome is regenerated
16
Q
Termination
7.1
A
- Elongation continues until stop codon (UAA, UAG or UGA) is translocated into the A site
- Release factors recognise these signals + terminate protein synthesis
- Prokaryotic: *RF1 recognises UAA or UAG, *RF2 recognises UAA or UGA
- Eukaryotic: *eRF1 recognises all 3 stop codons
17
Q
What are the biding sites of ribosomes?
7.1
A
They have 3:
- P (peptidyl)
- A (aminoacyl)
- E (exit)
18
Q
What is a polyribosome/polysome?
7.1
A
- *A group of ribosomes bound to an mRNA molecule.
- Once a ribosome has moved away from the initiation site, another can bind to the mRNA + begin synthesis.
19
Q
Translation regulation
7.1
A
- Transcription = main level of control of gene expression.
- Regulation of translation of *specific mRNA important in modulating gene expression
- Translation repressor proteins
- non coding micro-RNAs
- Translation can be *globally repressed in cellular stress, e.g. starvation, depletion of growth factors, or DNA damage
- Modulation of elF-2 + elF4E activity
20
Q
- Translational repressor bound to 3’ untranslated regions
7. 1
A
- Translational repressors can bind to regulatory sequences of 3’ untranslated region (UTR) + inhibit translation by binding to initiation factor elF4E, attached to 5’ cap.
- This interferes with translation by blocking binding of elF4E to elF4G
21
Q
- Regulation of ferritin translation (protein that stores iron) by repressor proteins
7. 1
A
When iron is absent, iron regulatory protein (IRP) binds to an iron response element (IRE) in 5’ UTR, blocking translation.
22
Q
Translation regulation by modification of initiation factors
7.1
A
- results in global effects on overal translation activity rather than specific mRNAs
- *phosphorylation of elF2 + leF2B by regulatory protein kinases bock exchange bound GDP for GTP, inhibiting initiation of translation
23
Q
- Regulation of elF4E
7. 1
A
- In absence of growth factors, elF4E-binding proteins (4E-BP) bind to this factor + prevent association with elF4G, inhibiting translation.
- Stimulation by growth factors induces phosphorylation of 4E-BP, which dissociated from elF4E + allows translation to begin.
24
Q
Protein folding + processing
7.2
A
- To be active polypeptides must *fold into characteristic 3 dimensional conformations
- *several polypeptide chains aggregate to form a functional complex
- Many undergo *additional modifications; cleavage, covalent union of carbohydrates + lipids, necessary for the correct function + localisation
25
Q
- Chaperones + protein folding
7. 2
A
- Chaperones: proteins that facilitate the folding of other proteins or assembly processes of complexes involving proteins
- Do not provide additional information for folding of proteins, it is determines exclusively by aa sequence (interaction between side chains)
- Bind to + stabilise unfolded or partially folded polypeptides, preventing folding or aggregation
- Chaperone types:
- bind to nascent polypeptide chains
- stabilised part. Folded polypeptides in transport to organelles
- involved in assembly of multiple polypeptide chains
- participate in assembly of macromolecular structures (e.g. nucleoplasmin)
26
Q
Role of chaperones
7.2
A
- A part. folded polypeptide is transported from the cytosol to mitochondria
- *Cytosolic chaperones: stabilise extended conformation
- *Mitochondrial chaperones: facilitate transport + subsequent folding of polypeptide chain within an organelle
27
Q
- Sequential actions of chaperones
7. 2
A
- Hsp70 chaperones + chaperonins found in both prokaryotic + eukaryotic cells
- *Hsp70 proteins stabilise polypeptide chains during translation + transport by binding to short hydrophobic segments
- Polypeptide then transferred to *chaperonin, where folding occurs.
- Chaperonins consist of subunits arranged in 2 stacked rings to form double-chambered structure. This isolates protein from cytosol + other unfolded proteins
28
Q
*How + which enzymes catalyse protein folding?
A
- Do so by breaking and forming covalent bonds
- *Protein disulfide isomerise (PDI): catalyse disulfide bond formation. PDI is abundant in ER where an oxidising environment allows (S—S) linkages.
- Peptidyl prolyl isomerase: catalyses isomerisation of peptide bonds involving proline residues. Isomerisation between cis + trans configurations of prolyl-peptide bonds could otherwise be rate-limiting step in protein folding
29
Q
- Proteolysis
7. 2
A
Important stage in maturation of many proteins
- *removal of initial methionine: from amino terminus of many polypeptides. Occurs at beginning of translation
- *New chemical groups: often added to amino terminus, e.g. acetyl or fatty acid chains
- *Elimination of amino-terminal signal sequence: in secretory proteins or membrane proteins in eukaryotic cells (see unit 8)
- Formation of functionally active proteins *cleavage of larger inactive precursors.
- *insulin: synthesised as precursor polypeptide that undergoes 2 cleavages to produce mature insulin
30
Q
- Glycosylation
7. 2
A
- Addition of carbohydrate chains to form *glycoproteins
- more frequent in eukaryotic cells. Normally secreted or located on cell surface
- *Functions:
- Protein folding in ER
- Targeting proteins to intracellular compartment
- Recognistion in intercellular interactions
- Types of glycoproteins (depen on binding site of carb.):
- *N-linked: carbohydrate attached to *nitrogen atom in side chain of *asparagine
- *O-linked: carbohydrate attached to *oxygen atom in side chain of *serine or *threonine
31
Q
Synthesis of N-linked glycoproteins
7.2
A
- Glycosylation starts in ER before translation completed
- Ogliosaccharide is assembled on a lipid carrier (dolichol phosphate) in ER membrane, then transferred to asparagine residue
- Further modifications result in many diff. N-linked oligosaccharides
32
Q
Synthesis of O-linked glycoproteins
7.2
A
- O-linked oligosaccharides are added to proteins in Golgi apparatus
- Formed by addition of one sugar at a time
33
Q
- Lipid binding
7. 2
A
- Often mark + anchor proteins to plasma membrane, being lipid it’s hydrophobic in nature, it is inserted into the membrane
- Proteins anchored to *cyytosolic face of eukaryotic cell membrane:
- *N-myristoylation: Myristic acid (14C) binds to N-terminal Gly.
- *Prenilation: Prenyl group attached to sulfur in side chains of Cys near C-terminus
- *Palmitoylation: Palmitic acid (16C) added to sulfur in side chains of Cys residues
- Proteins anchored to *extracellular face of eukaryotic cell membrane:
- Adittion of *glycolipids to carboxyl terminal end of some proteins
34
Q
- Regulation by small molecules
7. 3
A
- Most enzymes regulated by conformation change that cause modifications in their catalytic activity. These changes are often produced by *non-covalent union of small molecules (aa,nt)
- *allosteric regulation: regulatory molecule binds to an enzyme site other than the active center, modifying protein’s conformation + affecting activity:
- Retrohinibition of metabolic pathways
- Transcription factors
- Regulation of tf such as eEF-1α by binding of GTP or GDP
35
Q
- Phosphorylation + other modifications
7. 3
A
- Most common + best studied covalent modification that regulates protein activity in response to environmental signals
- *Protein kinases: transfer phosphate groups from ATP to hydroxyl groups of side chains of serine, threonine or tyrosine. Often components of signal transduction pathways
- *Phosphatases: reverse phosphorylation, catalysing hydrolysis of phosphorylated aa
Other modifications:
- (*)acetylations of lysine residues
- (*)methylations of lysine + arginine
- (*)glycosation of serine + threonine
- (*)nitrosylation of cysteine residues
- peptide additions
- ubiquitylation
- sumoylation
36
Q
- Protein-protein interactions
7. 3
A
- Many proteins have subunits, interactions between them can regulate activity
- e.g. *cAMP-dependent protein kinase has 2 regulatory + 2 catalytic subunits in inactive form
- cAMP binds to regulatory subunits, inducing conformational change + dissociation of complex
- Free catalytic subunits are then enzymatically active protein kinases
- cAMP acts as allosteric regulator, by altering protein-protein interactions
37
Q
Protein degradation
7.4
A
- Amount of proteins in cell regulated not only by rate of synthesis but also rate of degradation
- Half-life of proteins is highly variable, from few minutes to several days
- Regulatory proteins, e.g. FTs, are rapidly degraded
- Other proteins break down in response to specific signals
- Defective or damaged proteins degraded by:
- Ubiquitin-protea some pathway
- Lysosomal proteolysis
38
Q
- Ubiquitin-proteasome pathway
7. 4
A
- Main route of *selective protein degradation in eukaryotes involves *ubiquitin as protein marker.
- Polyubiquitinated proteins recognised + degraded by large complex with multiple subunits + protease activity, the *proteasome
Many proteins that control fundamental cellular processes are targets for regulated ubiquitylation + proteolysis.
- *Example: cyclins that regulate progression through division cycle of eukaryotic cells
- Entry of cells into mitosis controlled (in part) by cyclin B, a regulatory subunit of Cdk1 protein kinase.
- Active *cyclinB-Cdk1 complex induces entry into mitosis
- Degradation of cyclin B by proteasome leads to inactivation of Cdk1 kinase, allowing cell to exit mitosis + return to interphase
39
Q
- Lysosomal proteolysis
7. 4
A
- Proteins degradation can occur in lysosomes: membrane enclosed organelles that contain digestive enzymes, incl. *proteases
- Lysosomes digest extracellular proteins taken up by *endocytosis, and take part in turnover of organelles + proteins.
- Containment of enzymes in lysosomes prevent uncontrolled degradation
- Proteins move into lysosomes by *autophagy: vesicles (autophagosomes) enclose small areas of cytoplasm or organelles then fuse with lysosomes
- Autophagy activated in nutrient starvation, allowing degradation of nonessential proteins + organelles to reutilise components
