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Medical Biochemistry and Genetics > Lecture 2/3 - Protein Synthesis > Flashcards

Lecture 2/3 - Protein Synthesis Flashcards

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1
Q

Important facts about proteins

A

1) ~44% of dry weight of the human body
2) ~5% of human caloric intake goes to protein synthesis
3) They catalyze most of the reactions in living organisms
4) Serve many roles (enzymatic, structural, etc.)

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2
Q

What is the Central Dogma?

A
  • RNA to DNA to protein
  • States that once “information” has been passed into protein, it is irreversible
  • Transfer of information from nucleic acid to nucleic acid or from nucleic acid to protein is interconvertible but from protein to protein or from protein to nucleic acid is not
  • Information = precise determination of sequence, either of bases or of AA residues
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3
Q

What are the start and stop codons?

A
  • Start codon - AUG (Methionine, KUG)
  • Stop codons:
    1) UAG - AMBER
    2) UAA - OCHRE
    3) UGA - OPAL
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4
Q

What are the characteristics of the genetic code?

A

1) Co-linear triplet code - codons consist of 3 nucleotides and are colinear due to anticodons
2) Nearly universal - variations in mitochondria, mycoplasma, ciliates (mitochondria have their own transcription and translation systems)
3) Degenerate/redundant - many of the codons code for the same AA (61 codons for 20 AA)
4) Non-overlapping - triplet codons are distinct and only code for one AA
5) Unpunctuated - although some codons are signals - there is nothing differentiating between each triplet

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5
Q

What are point mutations?

A

Point mutations - changes of single nucleotides (i.e. some hemoglobinopathy examples)

1) Silent mutation - mutation that doesn’t result in a change in the AA but is only silent at the level of the protein
2) Missense mutation - mutation that changes to a different AA (i.e. HbS = Sickle Cell anemia, mutant beta chain)
3) Nonsense mutation - changes to a stop codon (i.e. some beta thalassemias) –> proteins derived become shorter and often get little to no protein due to nonsense-mediated decay (NMD), which destroys mRNA which give rise to shortened proteins
4) Suppressor mutation - mutation from a stop codon to a sense codon (i.e. Hb Constant Spring, alpha chain) –> read through where the protein should have ended, resulting in a longer protein

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6
Q

What are frameshift mutations?

A

Frameshift mutations - insertions or deletions of numbers of nucleotides not divisible by three

  • Both can result in longer or shorter proteins depending on the location of the next stop codon
    1) Insertion - i.e. Hb Tak, beta chain
    2) Deletion - i.e. some other beta0 thalassemias
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7
Q

What are the contents of the eukaryotic ribosome structure?

A
  • S = sedimentation coefficient (not additive)
  • Overall ribosome = 80S and breaks into larger and shorter subunits –> 60S and 40S, respectively
  • 60S subunit gives rise to 5S, 5.8S and 28S RNA subunits
  • 40S subunit gives rise to a 18S RNA subunit
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8
Q

What are the contents of the prokaryotic ribosome structure?

A
  • S = sedimentation coefficient (not additive)
  • Overall ribosome = 70S and breaks into larger and shorter subunits –> 50S and 30S, respectively
  • 50S subunit gives rise to 5S and 23S RNA subunits
  • 30S subunit gives rise to a 16S RNA
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9
Q

How many (1) antibiotics, (2) antifungals and (3) antivirals affect the process of translation?

A

1) About half - affecting prokaryotic translation NOT eukaryotic translation
2) None b/c fungi have similar translation system to eukaryotes
3) None b/c viruses use the host cells translation machinery for translation of their own mRNA

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10
Q

1) What is peptidyl transferase made of?

2) How long is the polypeptide exit tunnel?

A

1) The peptidyl transferase center consists of RNAs and is not proteinaceous
2) The exit tunnel is 40-50 AA long and buries the polypeptide until the protein becomes too large to fit within it

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11
Q

What are the differences between eukaryotic and prokaryotic mRNA?

A

1) Eukaryotic are mostly monocistronic (spliced) - containing one coding region vs polycistronic - containing more than 1 coding region - for prokaryotes
2) 5’ end:
* eukaryotes typically is capped = 7-MeGpppGXY and the cap is recognized by initiation factors
* prokaryotic factors are not capped and made of ppp
3) 3’ end:
* eukaryotes contain a poly adenylated tail (added post-transcriptionally)
* prokaryotes only have an OH group b/c there is no post translational modification
4) Eukaryotic mRNA is functionally circular - increases stability and translational efficiency due to machinery being closer together

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12
Q

tRNA structure

A

1) All are around ~70 nucleotides long and follow a cloverleaf structure with a 3’ extension = CCA
2) AA accepting end of the tRNA is located at the 3’ end
3) Carry “activated” AAs

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13
Q

Mupirocin

A

Topical antibiotic which affects Isoleucine tRNA synthase in bacteria

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14
Q

Aminoacylation of tRNA

A

1) AA + tRNA + ATP (aminoacyl-tRNA synthetase) AA~tRNA + AMP + PPi
* deltaG = 0 Kcal/mole
* AA is first activated by reacting with ATP generating aminoacyl-AMP
* The activated AA is then transferred from aminoacyl-AMP to tRNA at the 3’ end CCA

2) PPi + H2O (pyrophosphatase) 2Pi
* deltaG = -6.6 Kcal/mole

  • reaction driven by sequential linkage
  • Overall free energy change for aminoacylation of tRNA is -6.6 Kcal/mole
  • Enzymes are vital for the fidelity of protein synthesis: 2 steps allow “proofreading”
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15
Q

Wobble pairing

A
  • There are less tRNA species than there are codons for AA (50 vs 61) so it allows for different type of base pairing other than the regular Watson and Crick pairing, to alleviate tension. These include:
    1) G-C, but also G-U
    2) U-A, but also U-G
    3) I (hypoxanthine)-C , but also I-U and I-A
    • I is a modified G
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16
Q

Translation factors - Initiation (1 Prokaryotes, 2 Eukaryotes)

A
  1. IF1-IF3
  2. eIF1-eIF5 (>12)
    * IF = initiation factors
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17
Q

Translation factors - Elongation (1 Prokaryotes, 2 Eukaryotes)

A
  1. EF-Tu/Ts (EF1A, B) and EF-G (EF2)
  2. eEF1 and eEF2
    * Elongation factors
    * EF-G has GTPase activity
18
Q

Translation factors - Termination (1 Prokaryotes, 2 Eukaryotes)

A
  1. RF1-RF3
  2. eRF1, eRF3
    * RF = release factors
19
Q

How does the prokaryotic ribosome find its initiation site?

A
  • AUG start codon has a Shine-Dalgarno box upstream of it
  • There is a specific sequence at the S-D box which the 3’ end of the 16S rRNA of the 30S subunit looks to bind to and locks it near the start codon
  • Since it is polycistronic, there will be multiple AUG sequences and multiple S-D boxes
20
Q

How does the eukaryotic ribosome find its initiation site?

A
  1. Chief mechanism = Cap-dependent scanning - recognition of the 5’ cap by the 40S subunit allows for it to bind to initiation factors –> the 40S subunit will then scan down the untranslated region of the mRNA in a 5’ to 3’ direction (can scan over secondary structures) to find the correct AUG sequence and then stops
  2. Internal ribosome entry site (IRES) - elaborate structure of the 5’ end of the mRNA which would otherwise take too long to scan to find the start codon –> 40S subunit binds directly to the AUG site
21
Q

How does Streptomycin work?

A

It binds nucleic acids and messes up base pairing involved in the 30S-S/D box interaction, as well as messing up elongation and the ability to recognize the appropriate tRNA in prokaryotes
*It is an aminoglycoside which also causes miscoding during elongation

22
Q

Describe the process of elongation

A

1) AA-tRNA binding: methionine-carrying tRNA starts out in the P site. Next to it, a fresh codon is exposed in another slot, called the A site. The A site will be the “landing site” for the next tRNA, one whose anticodon is a perfect (complementary) match for the exposed codon
2) Peptidyl transfer: once the matching tRNA has landed in the A site, the formation of the peptide bond that connects one AA from the tRNA in the P site, onto the AA from the tRNA in the A site. The methionine forms the N-terminus of the polypeptide, and the other amino acid is the C-terminus.
3) Translocation: once the peptide bond is formed, the mRNA is pulled onward through the ribosome by exactly one codon. This shift allows the first, empty tRNA to drift out via the E (“exit”) site. It also exposes a new codon in the A site, so the whole cycle can repeat

23
Q

1) What is the characteristic feature of Diphtheria toxin?

2) What animal is the antitoxin raised in?

A

1) Thick gray coating (pseudomembrane)/leathery appearing throat
2) Horses - must run skin test for horse serum sensitivity prior to administering the antitoxin

24
Q

What is diphtheria toxin produced by?

A

Corynebacterium diphtheriae tox+ strains (phage)

25
Q

How does diphtheria toxin work?

A

*It is a protein that is cleaved into two fragments:
-Fragment B causes the toxin to be internalized into the eukaryotic cell
-Fragment A poisons translation - 1 molecule is sufficient to kill a cell
*It catalyzes ADP-ribosylation of eEF2 - transfer of ADP-ribose from NAD onto a modified histidine residue (called diphthamide) in eEF2
eEF2 + NAD+ –> ADP-ribosyl-eEF2 + nicotinamide + H+
*once the eEF2 is taken out of the eukaryote, then translation stops and the cell dies

26
Q

What is Ricin?

A
  • protein toxin from castor beans
  • <1 mg can kill an adult, 1 bean can kill a child
  • Consists of 2 polypeptide chains:
    • B chain binds a cell surface receptor for uptake
    • A chain depurinates 28S rRNA at a specific A residue (inactivating elongation)
  • This bean killed Georgi Markov
  • Abrin is a bean which is 75x more deadly
27
Q

Describe the process of translational termination

A
  • Occurs when a stop codon in the mRNA (UAA, UAG, or UGA) enters the A site
  • Stop codons are recognized by release factors, which fit neatly into the A site (though they aren’t tRNAs).
  • Release factors mess with the enzyme that normally forms peptide bonds: they make it add a water molecule to the last amino acid of the chain –> this reaction separates the chain from the tRNA, and the newly made protein is released.
  • After the small and large ribosomal subunits separate from the mRNA and from each other, each element can (and usually quickly does) take part in another round of translation.
28
Q

Translation controls: Translational Repression

A
  • Ferritin is an intracellular Fe2+ binding protein which is needed when Fe2+ in cells is at a high concentration
  • Ferritin synthesis is controlled at the level of translation
  • Iron response element (IRE) bind IRE-binding protein (IRE-BP), except when Fe2+ levels are high
    • IRE located at the 5’ end of ferritin
  • Ferritin mRNA translation is blocked by bound IRE-BP
  • IRE/IRE-BP binding also controls translation of eALAS mRNA and degradation of the transferrin receptor (TfR) mRNA
29
Q

Hereditary hyperferritinemia - cataract syndrome (HHCS)

A
  • IRE mutations in ferritin mRNA cannot bind IRE-BP leading to ferritin synthesis being constitutively on
    1) ferritin synthesis is increased
    2) ferritin released into serum (supposed to be intracellular)
    3) early-onset cataract formation
30
Q

Translation controls: Down regulation of supply of initiator Met-tRNAi via eIF2 kinases

A
  • eIF2 supplies Met-tRNAi to 40S subunit as ternary complex with GTP
  • eIF2 phosphorylation inhibits initiation by eIF2B

eIF2GDP –> (eIF2B) eIF2GTP –> eIF2GTPMet-tRNAi –> protein synthesis

*eIF2 kinases will phosphorylate eIF2 before it attaches to the GTP and when the phosphorylated eIF2 interacts with the eIF2B it will trap the eIF2B causing initiation to be inhibited

31
Q

Examples of eIF2 kinases

A
  • All respond to different kinds of stress
    1) HRI - reticulocytes minus heme
    2) PKR - interferon plus virus infection (dsRNA)
    3) PERK - ER stress
    4) GCN2 - AA starvation
32
Q

What causes Vanishing White Matter (VWM)?

A
  • Caused by a mutation in any of the 5 subunits of eIF2B –> reduced eIF2b level or function
  • Patients are born with the disease but neurological deterioration is usually exacerbated by head trauma
  • Variable severity and progression, autosomal recessive
33
Q

What are the symptoms of vanishing white matter?

A

1) Ataxia

2) Ovarian failure

34
Q

What does the (e)EF1 factor do?

A
  • Supplies aa-tRNA to ribosome during elongation
  • GTP/GDP exchange during elongation - hydrolysis of GTP is required for every elongation step
  • Similar GTP/GDP exchange cycle for eIF2

EF-TuGDP –> (EF-Ts) –> EF-TuGTP –> EF-TuGTPaa-tRNA –> protein synthesis

35
Q

Translational controls: Up regulation of mRNA binding via eIF4E

A
  • Most common form of regulation
  • eIF4E binds mRNA 5’ cap and is part of a complex required for scanning
  • Its function is increased by phosphorylation (kinase cascades) of (a) 4E-BP or (b) eIF4E itself
    a) Phosphorylation of 4E-BP releases eIF4E and rendering it more active into the other complex b/c when eIF4E is bound to 4E-BP it is not active
36
Q

Inhibitors of Protein synthesis: Streptomycin (Gentamicin, Kanamycin, Neomycin, etc)

1) Class
2) Target
3) Action

A

1) Aminoglycoside
2) 30S
3) Inhibits initiation and causes misreading

37
Q

Inhibitors of Protein synthesis: Tetracycline (doxycycline)

1) Class
2) Target
3) Action

A

1) Tetracycline
2) 30S
3) Inhibits binding of AA-tRNA to A-site

38
Q

Inhibitors of Protein synthesis: Chloramphenicol

1) Class
2) Target
3) Action

A

1) …
2) 50S
3) Inhibit peptidyl transferase

39
Q

Inhibitors of Protein synthesis: Erythromycin (Clarithromycin, azithromycin)

1) Class
2) Target
3) Action

A

1) Macrolides
2) 50S
3) Inhibit translocation

40
Q

Inhibitors of Protein synthesis: Puromycin

1) Target
2) Action

A

1) 50S, 60S

2) Premature release of nascent polypeptide

41
Q

Inhibitors of Protein synthesis: Diphtheria toxin

1) Target
2) Action

A

1) eEF2

2) Inhibits translocation (ADP ribosylation)

42
Q

Inhibitors of Protein synthesis: Ricin (from castor beans)

1) Target
2) Action

A

1) 60S

2) Inhibits binding of AA-tRNA to A-site (28S rRNA depurination)

Medical Biochemistry and Genetics (4 decks)

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