Lecture 8 Slides Flashcards
What does 45S precursor rRNA break into after chemical modification and cleavage?
18S rRNA, 5.8S rRNA, 28S rRNA
What precursor rRNA is used to make two ribosomal subunits? How many nucleotides does it have?
455 precursor rRNA
13,000 nucleotides
What translates rRNA
RNA polymerase III
Which piece is incorporated into small ribosomal unit
18S rRNA
Which pieces are incorporated into large ribosomal unit
5.8S, 28S and 5S, which is not made from 45S
How is 45S rRNA precursor modified? What are resulting modifications?
Post transcriptional modification of bases by snoRNPs
Pseudouridine and 2’-O-methylated nucleotide
How are ribosomal RNA genes arranged
Tandemly repeated and packed with transcripts /nucleolus
What does nucleolus contain
Tandem repeats of rRNA genes from several chromosomal loci
When does nucleolus reform
In each cll cycle
What is at edge of nucleolus, on nuclear envelope
Peripheral heterochromatin
Pathway for assembly of ribosomal proteins
rRNA gene transcribed to 45S precursor
SnoRNAs come in and modify and process rRNA
Ribosomal proteins made in cytoplasm come into nucleolus
5S rRNA joins in from cytoplasm
RNAs and proteins involved in rRNA processing are constantly recycled
Immature large subunit assembled
Telomerase proteins enters nucleus from cytoplasm and form telomerase with telomerase RNA
Small subunit is built
Large or 60S subunit exits nucleus
Small or 49S subunit exits nucleus
Form ribososome for translation
How many amino acids does genetic code specify
20
In what directions are codons presented
5’-3’
What does redundancy mean
64 codons specify 20 amino acids
What do stop codons specify in some mitochondria
Amino acids
What do stop codons specify in some protozoan nuclear genes
Amino acids
What unusual amino acid is specified by stop codon in some cells
Selenocysteine
What sets correct reading frame
What does it specify in eukaryotes? Prokaryotes?
AUG start codon
Methionine in eukaryotes
Formyl methionine in prokaryotes
What do tRNAs do
Translate codons to amino acids
What synthesizes tRNAs
RNA polymerase III
Extensively modified before exported to cytoplasm
What does adaptation of nucleotide sequence to amino acid sequence require
- Aminoacyl tRNA synthetase couples amino acids to appropriate tRNA, resulting in an aminoacy-tRNA or “charged” tRNA
- TRNA anticodon pairs with mRNA codon
What does charged tRNA look like
Cross with attached amino acid at 3’ end
Anticodon at bottom loop
D loop on left
T loop on right
What does linkage of amino acid to tRNA require energy wise
ATP
Wobble baee pairing
Some tRNAs pair with more than one codon
Can amino acids have more than one tRNA
Yes, with different anticodons
Bacteria wobble bases and possible anticodon bases
U. A,G, I
C. G, I
A. U,I
G. C,U
Wobble codon base for eukaryotes and possible anticodon bases
U. G,I
C. G,I
A. U
G. C
Prokaryotic ribosomes are composed of
50s and 30s combine to make 70s
50s molecular weight, parts and number of proteins
MW 1,600,000
5S rRNA and 23S rRNA
34 proteins
30S molecular weight, parts and protein no,
900,000 MW
16S rRNA
21 proteins
What makes up eukaryotic ribosome
60S and 40S combine to make 80S
60S molecular weight, parts and protein number
2,800,000 MW
5S and 28S
49 proteins
40s MW, parts, proteins
1,400,000 MW
18S
33 proteins
70s molecular weight
2,500,000 MW
80s molecular weight
4,200,000 MW
What are archaeal ribosomes composed of? What are their primary sequences closest to?
30S and 50S subunits join to make a 70S particle
Primary sequences of both RNA and protein components are closer to eukaryotes than to those of prokaryotes
What binds mRNA
Ribosomes
How many sites in ribosome for tRNA binding
What are they
Three
E, P, A
What kind of enzyme is ribosome
Ribozyme
What forms catalytic site for peptidyl transferase
RNA in large subunit
Where does tRNA add amino acid
To the C-terminal end of the growing peptide chain
Peptidyl transferase reaction
When activated tRNA adds to the C-terminal end of growing peptide
How does GTPase molecular switch work
Protein conformation changes when bound to GTP vs GDP, thus affecting activity if interacting proteins
What protein promotes exchange of GFP for GTP
Guanine nucleotide exchange factor (GEF)
What helper protein promotes hydrolysis of GTP
GAP
Is GTPase a protein kinase
No. No phosphate group is transferred
What is first amino acid for eukaryotes
Methionine
Where does initiator tRNA bind
P site of small subunit
How does small subunit bind mRNA
It binds 5’ cap and begins to scan for first AUG
Kozak sequence
AUG surrounded by sequence that indicates translation initiation point in eukaryotes
What sequences on prokaryote mRNA bind small subunit at AUG codon
Shine-Dalgarno sequences
Where does second charged tRNA bind
A site
What happens when small subunit finds first AUG
It causes eIF2 to hydrolyze GTP and large subunit binds
EF-Tu (EF1) role
Elongation factor. Checks accuracy of base pairing.
What happens of base is incorrectly paired
TRNA dissociates
EF-G (EF2)
Monitors translocation of ribosome along mRNA
What does switch helix do
EF-Tu undergoes conformational change after GTP hydrolysis. Switch helix in domain 1 allows domains 2 and 3 to rotate as a single unit to release tRNA
EF-Tu eukaryote equivalent
EF1
EF-G eukaryote equivalent
EF2
Stop codons are
UAA
UAG
UGA
Are there tRNAs for stop codons
No
Release factors
Proteins that mimic tRNAs, bind in A site
What does peptidyl transferase do at termination of translation
Catalyzes addition of water molecule to C-terminus. Peptide is released from ribosome.
What is a polyribosome
Multiple ribosomes working on mRNA at once
How does ribosome know if protein is destined for endoplasmic reticulum
A signal at N-terminal
Benefits of circular structure of polyribosome
Facilitates structure of polyribosome
Stabilizes mRNA
What determines whether ribosome is free or membrane bound
Information in nascent polypeptide
What determines the energetically favored final 3d structure of a
protein sequence of amino acids
Which proteins can reach folded state independently
Small proteins <100 aa
What do larger proteins need to fold in cytosol
Chaperone proteins for folding in cytosol’s high protein concentration
What does accumulation of misfiled proteins cause
Impaired cellular function
What are some human diseases that involve miss folded proteins. (5)
Alzheimer's Parkinson's Cystic fibrosis Jacob-Creutzfeldt disease Inflammation
Three ways folding of protein can go
On pathway folding
Off pathway folding
Irretrievable accidents
What fixes protein if it has misfolded
Chaperone catalysis
What happens if misfolded can’t be fixed
Digested by proteases
Role of ribosome bound chaperone systems
Help proteins fold as they exit the ribosome
How large is ribosomal tunnel
1-2 nm in diameter
What ahopens through ribosomal tunnel
Unfolded polypeptide chains or alpha helices pass through. Peptides that are about 40 aa are contained within tunnel
Role of trigger factor in bacteria
Its activity folds about 70% of proteins
What folds bacterial protein besides tigger factor activity
Hsp60 and hsp70
How does trigger factor work
It forms a cavity that protects and folds nascent protein chains of ~60 amino acids
Role of chaperone systems
Facilitate protein folding
Which cells do not have trigger factors
Archeal and eukaryotic
Ribosome bound chaperones in archaeal and eukaryotic cells
NAC complex and RAC complex (hsp70 homolog). Play same role of folding nascent polypeptides.
PFD
Prefoldins bind a subset of proteins including the cytoskeletal proteins actins, alpha-tubulin, beta-tubulin
Brings client proteins to chaperonin complex (hsp60) for folding
NAC
Nascent polypeptide-associated complex
RAC
Ribosome-associated complex, an hsp70 homolog
How are nascent proteins recognized for the ER
Signal recognition particle (SRP)
Role of Cytosolic chaperone system (hsp70 system)
Constitutive - carries out routine protein folding
Stress induced- accumulates rapidly when cells experience increased temperatures (heat shock) or other stresses
What happens if protein is incompletely folded?
May be further folded by another round of hsp70, passed on the hsp60 or hsp90 chaperone
When is folding accomplished
When hsp70 shields hydrophobic regions in the client protein
Hsp60
Cytosolic chaperone system
Hsp60 family chaperones form a barrel structure; unfolded protein enters to be folded
What does unfolded protein bind in hsp60. Folding process?
Hydrophilic residues exposed in GroEL surface
ATP and GroES bind
Expose hydrophilic residues in chamber, which drives protein folding
How is hsp60 structured
Two identical stacked rings, each containing eight different proteins
Complex is 800 KD
Cycle is ATP regulated, but folding is accomplished by cycles of protein encapsulation
What makes up chaperonin family
Tcp1 in cytosol of eukaryotic cells
Hsp60 in mitochondria
GroEL and GroES in bacteria
