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Biology > Unit 7: Protein Synthesis, Processing And Regulation > Flashcards

Unit 7: Protein Synthesis, Processing And Regulation Flashcards

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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)
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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
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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
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4
Q
  • What are tRNA synthetases?

7. 1

A

Selective enzymes that attach appropriate aa onto its corresponding tRNA

Mechanism summarised into following reactions:

  1. Amino Acid + ATP => Aminoacyl-AMP + PPi
  2. Aminoacyl-AMP + tRNA => Aminoacyl-tRNA + AMP
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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)
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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)

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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)
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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
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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)
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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
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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
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12
Q

Steps of initiation of translation (bacteria)

7.1

A
  1. 30S ribosomal subunit binds IF1 + IF3.
  2. MRNA + initiator N-formylmethionyl (fMet) tRNA + IF2 (bound to GTP) join complex.
  3. TRNA binds to start codon. IF1 + IF3 released.
  4. 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.

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13
Q

Steps of initiation of translation (eukaryotes)

7.1

A
  1. ElF1, elF1A + elF3 bind to 40S subunit.
  2. ElF2 bound to GTP, binds to initiator methionyl tRNA.
  3. Pre-initiation complex formed by associating all + elF5.
  4. 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).
  5. Factors direct mRNA to 40S subunit through interactions between elF-4G + elF-3.
  6. 40S subunit bound to methionyl tRNA + elF checks mRNA until AUG start codon identified. Displacement requires energy.
  7. 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.
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14
Q

Steps of elongation

7.1

A
  1. Initiator methionyl tRNA binds to P site.
  2. Elongation factor (EF-Tu in prokaryotes, eEF1α in eukaryotes) complexed to GTP brings aminoacyl tRNA to ribosome.
  3. Next aminoacyl tRNA binds to A site by pairing with 2nd codon of mRNA. Hydrolysis of GTP + release of EF.
  4. Peptide bond is formed, catalysed by large ribosomal subunit. Initiator TRNA (uncharged) now at P site.
  5. 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).
  6. When next aminoacyl tRNA binds to site A, free tRNA from site E released.
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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
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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

Biology (11 decks)

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