L14 : The Ribosome Flashcards
How are ribosomes efficient?
Rate of translation = 10-20 AA per sec (1-3 AAs in eukaryotes)
Multiple ribosomes can translate from 1 mRNA transcript
Explain coupling of transcription and translation in prokaryotes?
Genes of similar function placed within same operon (promoter) so multiple proteins from same mRNA transcript
Precise adjustment of transcription in relation to translation
Explain coupling of transcription and translation in eukaryotes?
Occur in separate compartments with generally 1 protein product per mRNA transcript
Spatial and temporal separation of processes assists expression regulation (mRNA processing, splicing, joining of exons, 5’ capping/polA tailing)
What is the structure of the peptidyl transferase centre?
Made up of tRNA binding sites that are common to both subunits
A-site = binds incoming aminoacyl tRNA
P-site = contains polypeptide chain
E-site = exit site of deacetylated tRNAs
What is the basic structure of the 70S ribosome subunit?
50S + 30S
2/3 rRNA
A,P,E sites
Helical structure
What is the structure of tRNAs?
17-20 kDa
Acceptor stem binds AA
T arm binds elongation factor
Anticodon loop recognises and binds complementary mRNA codon
Explain codon usage?
61 tRNAs for 20 AAs
Overabundance due to redundancy, though preferences exist
Linked to translational control where ‘rare’ codons slow translation
What is the structure and function of 30S subunit?
0.8 MDa
21 proteins and 16S rRNA (~1500 nucleotides)
Initiates binding to mRNA at Shine-Dalgarno sequence (aligns start codon)
Initiation factors can then assist in assembling initiation complex
Ensures correct translation via decoding region (antibiotics)
What is the structure and function of 50S subunit?
1.5 MDa
34 proteins and 5S/23S rRNA (~3000 nucleotides)
Contains peptidyl transferase centre (domain V) and ribosomal exit tunnel
Peptide bond formation typically occurs here
What is the A-minor motif?
A (often buried in rRNA) forms structural motif for stabalising 23S rRNA structure
4 different types
Specific H-bonding network between 2 RNA strands defined by position of 2’OH
What are the properties of ribosomal proteins?
55 proteins in 70S subunit
Generally 1 or more globular domains (surface)
Long N- or C- terminal extensions (buried)
Recognise RNA through shape and charge complementarily
How is the complex stabilised?
12 intersubunit interactions (mainly RNA-RNA) hold 70S together
Stabilised by Mg2+ ions at core, mediating overall function of ribosome
Are all ribosomes from different organisms the same?
Eukaryotic ribosomes are larger and have more expansion elements, likely to increase complexity of translation regulation
Ribosomal core is highly conserved across prokaryotes and eukaryotes
Can also consider mit vs cyt
What is the role of IF, EF, TF in translation?
Initiation factors - assembly of ribosome onto mRNA
Elongation factors - Permit elongation fo polypeptide chain
Termination factors - end translation, release protein, disassembly
What is the role of EF-Tu and EF-G?
EF-Tu delivers aminoacyl tRNA to ribosome
EF-G promotes translocation of tRNAs during synthesis
What is the function and model of the stalk region?
Major binding site for ribosomal factors (highly mobile)
bL 12 stalk and GTPase associated centre
Model from archaeal ribosome:
bL12 forms complex with L10 and L11
H43/H44 helices form platform upon which L10 and L11 bind
bL12 exists as dimer - identical proteins but L7 is acetylated
What is the location and dimensions of the ribosomal exit tunnel?
Located in 50S subunit
100A in length, 20A at widest
Holds 30-35 AA NC in extended conformation
Lined with 23S rRNA, constricted by loops uL4/uL22, shaped by uL23/uL24
What are the local regions of the exit tunnel and their folding events?
Upper/central - Narrow, can support formation of turns (eg. alpha helices)
Lower - Wider, allows more rudimentary structures to form (eg. beta hairpin)
Exit vestibule permits limited folding
What is the mechanism of arrest motifs?
AAs within certain motifs may interact with rRNA
Relayed back to PTC, preventing incoming tRNAs and stalling elongation
Eg. SecM (17 AA)
Explain bifurcation within exit tunnel?
Bifurcation occurs in lower half (around uL23)
NC interactions with ribosomal tunnel modulate timely folding events and exposure to chaperones
NC pathway dictated by sequence and length
Short NCs with unfolded segments directed to pathway 2, with H6/H7 rRNA
How does the ribosome ensure rapid and accurate translation?
Recognition of appropriate tRNAs
Maintaining reading code during translation
Decoding codon information to an appropriate tRNA (must be cognate)
Wobble effect - degeneracy in 3rd but not 1st or 2nd
What is the general mechanism for decoding?
Decoding centre of 30S:
16S rRNA, G530 of 530 loop (helix 18) and A1492/1493 (helix 44)
When correct codon binds, 530 pseudoknot form in 16S rRNA
Changes conformation to interact with minor groove of codon-anticodon double helix
Senses shape of WC bp in position 1 and 2 but not 3
What is the structural basis for decoding?
Codon sampling:
AA-tRNA complex delivered to PTC by EF-Tu*GTP
Anticodon on tRNA samples A site to verify match with mRNA codon
Codon recognition:
When cognate codon binds, conformational change in decoding centre seen, stabilising tRNA in A site
G530 and A1492/1493 residues interact primarily with minor groove of codon-anticodon helix, ensuring fidelity at the first two positions
Position 3 more tolerant of wobble base pairing, allowing degeneracy
GTP hydrolysis and EF-Tu domain shift:
30S subunit domain closure triggered, causing changes in loops of EF-Tu
His84 on EF-Tu exposed and enables catalysis of GTP hydrolysis to GDP
EF-Tu undergoes conformational change, 100 deg swing causing it’s dissociation
Accommodation:
tRNA shifts fully into A site ready for peptide bond formation
What are some examples of ribosome-associated proteins binding at exit tunnel base?
Trigger factor (TF) at uL23 to mediate folding
Signal recognition particle (SRP) at uL23/29 for translocation through membranes
Peptide deformylase at uL22 for removing formyl froup on start methionine (Nt processing)
How does TF (prokaryotes) assist in NC folding?
47 kDa protein, bindgs at L23
Cycles on/off ribosome with mean residence time of 11-15s
In presence of emerging NC, affinity for ribosome increases 9-30 fold and mean residence time increases to 50s
Forms a sort of ‘protective cage’ around NC, which threads through
What are advantages of ribosome acting as holdase?
Hands NC over to auxilliary proteins (eg. TF)
Protect NC against misfolding
Delays folding
Guides co-translational folding intermediates
How was the holdase activity of the ribosome investigated?
Using alpha-synuclein (intrinsically disordered protein) allowing study of RNC interactions without complications of folding
Contains charged residues and aromatics so can evaluate guiding interactions between ribosome and NC
Collected N15 NMR spectra for different NC complexes and compared to control of isolated syn + 70S
Contact is made approx 45 AA from exit tunnel
What is the ribosome holdase effect?
Neg charge of ribosome is counter balanced by Mg2+ ions, allowing transient interactions with NC
Pos charged NC able to make contacts with neg ribosome surface and then can interact with TF
What is the link between disease and ribosome dysfunction?
Composition of ribosome can be altered
Early development and disease linked to dysfunctional ribosome structure
Congential - bone marrow failure (Diamond-Blackfan anemia)
Somatic - cancers (blood/breast), mutations within ribosome can affect speed + fidelity
