Translation and Protein Processing Flashcards
The mRNA is translated __’ to __’ into protein (_’ to _’)
5’ to 3’
N’ to C’
STart codon
The first AUG in the open reading frame of an mRNA sequence; codes for the start methionine (first Met in the coding sequence)
How to stop codons work?
Stop codon: last codon of the mRNA open reading frame; UAA, UAG, UGA; does not encode an amino acid
Degeneracy of the genetic code
An amino acid can be coded by more than one codon (usually at the third position)
Frameshift mutation
Insertion or deletion of one or two dNTs, shifting/altering the ORF and changing all the amino acids downstream (3’) of the mutation
Silen tmutations
A point mutation that has no effect on the encoded amino acid because of redundancy in the genetic code’s third position (Wobble hypothesis)
Missense mutation
A POINT mutation that changes the amino acid of a single codon; probably changed the first or second position of the codon.
Nonsense mutaiton
Poin tmutation that changes a codon from one that encodes for an amino acid into a stop codon (UAA, UAG, UGA)
Structure of tRNA
Cloverleaf-shaped molecules read by RNA poly 3 that are post-transcriptionally modified to have a CCA sequence at their 3’ end
Activation of tRNAs
Activated at the 3’ end with an amino acid, forming an aminoacyl-tRNA complex. The tRNA anticodon hydrogen bonds w the mRNA codon.
What powers translation initiation factors?
GTP hydrolysis
Translation initiation process
- eIF3 binds to the 40S ribsomal subunit to maintain the 40S and 60S subunits separate
- eIF2 helps bind the initiation tRNAiMet to the 40S to form the preinitiation complex (ribosome+tRNAiMet)
- The mRNA of the eIF4F complex is brought to the 40S subunit
-
eIF4A unwinds the mRNA and eIF4B scans it to find the translation start site
- Initiation complex (ribosome+tRNAiMet+mRNA+eIF4F)
- 60S ribosomal subunit binds, eIF3 is lost
eIF3
eIF3 binds to the 40S ribsomal subunit to keep the 40S and 60S subunits separate
eIF2
eIF2 positions the aminoacyl-tRNAiMet over the 40S subunit at the P site to form the preinitiation complex (ribosome+tRNAiMet)
eIF4A & B
eIF4A unwinds the mRNA and eIF4B scans the mRNA to find the translation start site
A site of the ribosome
A site: where newly activated aminoacyl-tRNA molecules enter the ribosome, as dictated by the mRNA codon
(Blocked by tetracycline)
P site of the ribosome
Where the mRNA start codon and the tRNAiMet anticodon hydrogen bond
Where the growing peptide forms peptide bonds with the aa at the A-site
E-site
Exit site for tRNA molecules that have given up their amino acid.
Peptidyltransferase reaction of elongation
The a-amino group of the AA2 in the A-site attacks the a-carboxyl group of the amino acid (Met) in the P site, forming a peptide bond.
This breaks the bond between the Met and the 3’ end of the tRNAiMet in the P-site.
Now, the A-site holds a dipeptide and the P-site holds the deaacylated tRNAiMet
(Blocked by chloramphenicol)
translocation
The entire ribosome moves along the mRNA by one codon towards the 3’end of the mRNA, thus shifting the dipeptidyl-tRNA from the A site to the P-site and leaving the A site empty
Powered by the GTP hydrolysis
(Blocked by erythromycin)
Tetracycline
blocks binding of aminoacyl-tRNA to the A-site
Streptomycin
Prevents transition from initiation complex to chain elongaiton; also causes miscoding
Chloramphenicol
blocks peptidyl transferase reaction
Erythromycin
blocks translocation reaction
Rifamycin
Block stranscription initiation by binding RNA poly
Leukoencephalopathy with CNS hypomyelination and vanishing white matter
What causes it? What determines severity?
Missense mutation in eIF2 –> degeneraiton of white matter/loss of myelin during fevers –> neurological problems
The earlier age of onset, the more severe
How do mutations in eIF2 cause vanishign white matter?
eIF2 mediates formation of the preinitiation complex and stalls translation during fevers.
Thus, a missense mutation to eIF2 allows fever heat to denature proteins formed in translation –> misfolded proteins cause glial cell death
Describe the cleavage of the amyloid precursor protein (APP) by different secretase enzymes to the development of Alzheimer’s disease?
APP is normally cleaved by
- a-secretase (ADAM 10 or ADAM 17) cleaves in the extracellular domain
- y-secretase (Presenilin 1, PSEN 1) cleaves in the intracellular domain
In Alzheimers, a brain- specific B-secretase cleaves it in a different extracellular location while PSEN1 still cleaves in the intracellular domain. This generates a 42 residue peptide (AB42) secreted by neural and glial cells that forms the pathogenic B-amyloid plaque
What protein is misfolded in type II diabetes?
amylin/IAPP (prevents post-prandial spikes in blood glucose)
Results in apoptosis of B islet cells
What protein is misfolded in Alzheimer’s
Amyloid precursor proteins (APP) misfolded into AB42 (B-amyloid), resulting in neuronal death
What amylodi is formed in Parkinson’s disease?
a-synuclein is misfolded into lewy bodies, resulting in dopaminergic neuronal death
Familial Alzheimer’s (early onset) is associatd with what two genetic loci?
Mutations in the APP sequence cluster near AB42
Mutations within PSEN1 or 2
What amyloid is formed in prion disease?
PrPc is misfolded into PrPsc, resulting in neuronal death
What amyloid is formed in Huntington disease?
Huntingtin protein (MAP, vesicle trafficking) is misfolded into mHTT and trinculeotide repeats (polyQ), resulting in neurotransmitter release and neuronal death
What protein is misfolded in spinal cerebellar ataxia?
Ataxin is misfolded into trinucleotide repeats (polyQ) and nonpolyQ, resulting in neuronal cell death.
What protein is misfolded in ALS?
SOD (destroys oxygen radicals) –> dementia & neuronal atrophy
Three loci that contribute the development of early vs late onset Alzheimer’s
- Mutations within the APP sequence cluster near AB42 enhances proteolysis that lead to AB42 production
- Mutations within PSEN1 or 2 enhances and increases cleavage
- Allelic forms of ApoE (clears AB42)
- E4/E4 increases the risk of Alzheimer’s
- E2/E2 is protective
- E3 neither enhances or prevents
ricin structure & mechanism as poison
- A chain: globular peptide with the active site
- B chain: barbell-shaped; contains galactose binding sites
The two peptide chains are bonded by disulfide bonds and facilitate cell entry through hydrogen bonding to specific membrane sugars –> domain A depurinates adenines in the 28S rRNA to prevent translation
Late onset Alzheimer’s is associated with environmental factors AND
ApoE allelic forms
Kozak sequence
Site on mRNA where ribosome binds; include sthe start Met codon
Elongation release factors (eRFs)
Bind stop codon in the A-site, activate hydrolysis of the petpide chain from the tRNA in the P-site –> ribosome disassembles
molecular chaperones’ 3 functions
- Prevent inappropriate hydrophobic aggregations between nearby amino acids by binding to hydrophobic side chains
- Refolds denatured proteins
- Keeps peptides unfolded as membrane translocation occurs
Disuflide bonds form under reducing/oxidizing conditions. Where?
Disulfide bonds form under oxidizing conditions in the ER (NOT the cytosol, which is more reducing)
Peptidyl prolyl cis-trans isomerase (PPI)
converts prlines from trans to cis to form hairpin B-turns
Protein disulfide isomerase (PDI)
breaks inappropriate disulfide bonds and form new ones until native conformation is reached
Initially, the amyloid precursor protein (APP) is a-helical, but proteolytic cleavage causes it to misfold into a B-sheet that aggregates into _____ . Where?
Bamyloid plaques in the extracellular space between neural cells
Acylation and prenylation both occur on the cytosolic leaflet
Loss of Ras prenylation results in
decrease in Ras oncogenic transformation (good thing)
Ras prenylation inhibitors = cancer therapy
GPI-linked proteins occur on what leaflet of the membrane?
extracellular leaflet; common in lipid rafts, involved in blood coagulation, and signal transduction
Acetylation pharmacological significance
makes molecules more permeable to the blood-brain barrier
Ex) Aspirin
Methylation prevents
acetylation.
Calmodulin is a tri-methylated signaling molecule
Prothrombin time
- Take blood sample
- Mix with citrate, which will prevent clotting by binding Ca++
- Remove cells and add excess Ca++ to reverse the citrate inhibition, allow prothrombin to bind it, and time the clotting.
Phosphorylation
Addition of a negatively charged phosphate to the OH of a Ser, Thr, or Tyr.
Important in enzyme regulation and hormonal control
Casein
milk protein that contains many phosphoserines that bind Ca++. Thus, breast milk contains Ca++, phosphate, and amino acids.
Preproinsulin > proinsulin > insulin
- Cleave off the N’ end signaling sequence & form disulfide bonds –>proinsulin
- Cleave off an internal C-peptide –> Insulin (A chain & B chain held together by disulfide bonds)
Holoenzyme
Coenzyme with its prosthetic group
Apoenzyme / apoprotein
Inactive enzyme without its coenzyme prosthetic gorup
