How many reading frames are in dsDNA and mRNA?
6 dsDNA
3 mRNA
Open reading frame contains?
1) start codon
2) coding sequence
3) stop codon
Eukaryotic tRNA molecules transcribed by?
RNA pol. III
Eukaryotic tRNA molecules are processed….?
before leaving nucleus, trimed, and spliced (catalyzed by proteins)
What % of tRNA are modified bases?
10
Modified bases of tRNA molecules influence?
mol. conformation, base-pairing, amino acid coupling
Degenerate genetic code (numbers only)
4^3= 64 codons code for 20 amino acids
What are 2 possibilities that allows genetic code to be degenerate?
1) multiple tRNAs with multiple anticodons
2) 1 tRNA (with one anticodon) can regonize multiple codons
aminoacyl-tRNA synthetase
is a dual check mechanism that couples correct amino acid to cognate tRNA, where tRNA acts like activated carrier to shuttle activated amino acid
How does aminoacyl-tRNA synthetase assure correct amino acid pairing?
synthetase assesses nucleotide sequences of 1) anticodon, 2) amino acid acceptor arm, and 3) several other positions of tRNA
Where does translation occur?
ribosomes in the cytosol (E, P, A site)
Translation mechanism
1) small and large ribosomal subunits assembled at nucleolus
2) binding of mRNA joins subunits together to fomr a ribosome
Large ribosome subunit contains what?
peptidyl transferase activity
Peptidyl transferase activity mechanism
peptidyl-tRNA ttached to CTD of growing polypetide chaine
aminoacyl-tRNA frees tRNA molecules from peptidyl linkage
new peptidyl-tRNA molecule attached to CTD of growing polypetide chain
3 tRNA binding sites are?
Aminoacyl-tRNA
peptidyl-tRNA
Exit
How many tRNA binding sites are occupied at the same time?
2
EF-Tu proofreading mechanisms for improving translation fidelity?
1) tight codon-anticodon pairing
2) delay of peptidyl-residue transfer
Ef-Tu: tight codon-anticodon pairing
16S rRNA triggers conformational change in ribosome, EF-TU catalyzes GTP hydrolysis and dissociates from amino acyl group of tRNA
eF-tu: peptidyl-residue transfer delay
weakly bound tRNA in A site, will cause tRNA to dissociate before peptidyl residue can be transferred
EF-G
move ribosome forward
How do biological processes overcome limitations to complementary base-pairing?
induced fit, kinetic proofreading and the sequential se of both mechanisms
Why is translation initiation important?
determines reading frame and translation frequency
Translation initiation mechanism
1) MtRNA + eIFs bind to small rSU in P site
2) Small rSU binds to 5’ cap with bound eIF4 E + G
3) Small rSU searches for first (5’ located) AUG eIFs drives this movement via ATP hydrolysis)
4) Encounter of start codon releases eIFs and
large rSU binds
Bacterial translation initiation is mediated by?
Shine-Dalgarno sequence (AGGAGGU)
Shine-dalgarno
initiates bacterial translation:
functions as ribosome binding site through base pairing, replaces function of 5’ cap of eukaryotic mRNA, enables polycistronic mRNAs
translation termination mechanism
-Release factors bind to stop codon
- peptidyl transferase add
water to growing peptide chain to release peptide from a site
-ribosome disassembles
How is translation efficiency boosted?
polysomes (5’cap-poly A interaction allows efficient recycling of ribosomes)
What triggers mRNA degradation?
abnormal splicing causes ribosome to stall at stop codon in the presence of EJC
Co-translational protein folding
1) growing polypetide chain
2) folded N-terminal
3) folding CTD
4) folding of protein is complete after ribosomal release
Two types of heat shock proteins
HSP70
-HSP60 (chaperonin)
HSP70 bind to?
- bind to nascent peptide chain from ribosome
- hydrophobic patches
- bind tightly after ATP hydrolysis and dissociate after ATP rebinding
HSP60
form a barrel that acts like an isolation chamber, where the opening contains hydrophobic patches where misfolded proteins can be recognized
E3 ligase
recognizes unfolded proteins and marks them for degradation
proteosomes
degrade proteins into small peptides (nucleus and cytosol)
Proteosomes recognize?
substrates fused to L48 linked ubiquitin chain
Proteosome cap
unfoldase that unfolds proteins and threads them into proteasome
unfoldase
hexameric AAA protein sharing structural homology to helicases and dynein
Regulated destruction: What does it control and how can it be induced?
proteins
- activation of ubiquitin ligase
- activation of degradation signal
activation of ubiquitin ligase
1- protein kinase phosphorylates protein
2- allosteric transition caused by ligand binding
3- allosteric transition caused by adding protein subunit
activation of degradation signal
1-phosphorylation by protein kinase
2- unmasking by protein dissociation
3- destabilizing N-terminus
Where are small and large ribosomal subunits made?
nucleolus