Transcript Document

Translation
Initiation, Elongation, Termination
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Protein synthesis very complex
• Requires:
– Various tRNAs with their attached amino acids
– Ribosomes
– mRNA
– Numerous proteins with different functions
– Cations
– GTP
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Protein synthesis very complex
• Bacterial/eukaryotic translation similar
– eukaryotes need more nonribosomal proteins
– Both involve initiation, elongation & termination
• Ribosome starts translation at initiation
codon
– Establishes reading frame
– GUG can be initiator (f-met inserted here; valine
if codon is internal)
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Prok. Step 1: small ribosomal
subunit binds to mRNA
• Bacterial mRNAs have Shine-Dalgarno
(S-D) sequence
– 5 - 10 nucleotides before initiation codon;
– S-D complementary to 16S small subunit
sequence near 3' end
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Prok. Step 1: small ribosomal
subunit binds to mRNA
• AUG recognition by complementarity
• Initiation factors (IFs in prok; eIFs in euk) –
– prokaryotic cells require 3 initiation factors
– IF1, IF2, & IF3 bind 30S subunit & help it attach to
mRNA
– IF2 is GTP-binding, required for adding first
aminoacyl-tRNA
– IF3 may prevent the large (50S) subunit from joining
early
– IF1 may stop aa-tRNA from entering wrong site on
the ribosome
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Prok. Step 2: first aa-tRNA binds
to ribosome at AUG
• Methionine is always first amino acid
incorporated in protein chain
– In prokaryotes, formyl group (Nformylmethionine)
– Usually removed enzymatically; ~50% of time it
remains
– 2 distinct methionyl-tRNAs: tRNAiMet ; tRNAMet
– Mitochondria & chloroplasts initiate with Nformylmethionine
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.47
Prok. Step 2: first aa-tRNA binds
to ribosome at AUG
• Aminoacylated initiator tRNA enters the
preinitiation complex
– binds to both the AUG codon of mRNA & the IF2
initiation factor
– IF1 & IF3 are released
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Prok. Step 3: complete initiation
complex
• Large subunit joins
• GTP bound to IF2 is hydrolyzed
– GTP hydrolysis drives ribosome conformational
shift
– Causes release of IF2-GDP
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.47
Eukaryotic Initiation
• 10 initiation factors: 25 polypeptides in total
– Many (eIF1, eIF1A, eIF3) bind to 40S subunit
– prepares the subunit for binding to mRNA
• Initiator tRNAfMet also binds the 40S subunit prior to
its interaction with mRNA
– Initiator tRNA enters subunit in association with
eIF2-GTP, which is homologous to the bacterial
IF2-GTP
– Next, 43S preinitiation complex binds to 5' end of
mRNA
– methylguanosine cap aids in recognition
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.48
Eukaryotic Initiation
• mRNA already bound with initiation
factors
– eIF4E binds to 5' cap of eukaryotic
mRNA
– eIF4A moves along 5’end
• removes any double-stranded regions
– eIF4G links 5' capped end & 3'
polyadenylated end
• circularizes message (reason not clear)
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.48
Eukaryotic Initiation
• 43S complex scans to AUG
– Kozak consensus: 5'-CCACCAUGC-3'
– Then, eIF2-GTP is hydrolyzed
– eIF-GDP & other eIFs are released
– large 60S subunit joins the complex to
complete initiation
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
The Role of the Ribosome
• EM: highly irregular shape with bulges, lobes,
channels & bridges
• X-ray crystallographic studies (1990’s)
– 3 sites for association with tRNAs
– the sites receive each tRNA in successive steps
– A (aminoacyl) site – tRNA enters here (except
tRNAiMet)
– P (peptidyl) site - tRNAs donate aa of growing chain
– E (exit) site - tRNA leaves from here after losing aa
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.49
The Role of the Ribosome
• tRNAs span the gap between the 2
ribosomal subunits
– anticodon end of tRNAs contacts the small
subunit
– plays a key role in decoding the information
contained in the mRNA
– aa end of tRNAs contact the large subunit,
– plays a key role in catalyzing peptide bond
formation
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
The Role of the Ribosome
• Interface between small & large subunits
– spacious cavity lined almost exclusively by RNA
– small subunit facing cavity: single ds RNA helix
– bring together mRNA & incoming tRNAs
– primordial ribosomes made of RNA? (ribozymes)
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
The Role of the Ribosome
• Peptidase active site also largely RNA
– deep cleft
– protects peptide bond from hydrolysis
• A tunnel through the large subunit
– Begins at the active site
– Path of elongating polypeptide
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.49
Elongation
• Step 1: Aminoacyl-tRNA selection
– after initiation, initiator tRNA is in P site with A
site empty
– Second aminoacyl-tRNA enters A site
• tRNA already bound with EF-Tu (prok) or eEF1a (euk)
• EF-Tu associated with GTP
• rRNA small subunit verifies proper codon-anticodon
• Then, GTP is hydrolyzed
• Then, the Tu-GDP complex is released
• Regeneration of Tu-GTP from Tu-GDP requires EF-Ts
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Elongation
• Step 2: Peptide bond formation
– Amino group in A site reacts with carboxyl
group in P site
– P site tRNA no longer charged (deacylated)
– fMet transferred to dipeptide on tRNA in A
site
– No energy required
– Catalyzed by peptidyl transferase of large
subunit (ribozyme)
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Elongation
• Step 3: Translocation
– requires GTP-bound elongation factor & GTP
hydrolysis
• G (prokaryotes)
• eEF2 (eukaryotes)
– ribosome moves along mRNA in 5'—>3'
direction
• tRNA-dipeptide moves to P site
• deacylated tRNA moves from P to E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.50
Elongation
• Step 4: Releasing the deacylated tRNA
– Leaves E site
• Elongation cycle takes ~0.05 sec
– aa-tRNAs from cytosol probably rate limiting
– at least 2 molecules of GTP are hydrolyzed per
cycle–
• one during aminoacyl-tRNA selection
• one during translocation
– new aminoacyl-tRNA binds
• new peptide bond, etc.
– Elongation cycle continues until termination
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Elongation
• Reading frame and elongation
– Most destructive mutations are frameshift
mutations
– Some mRNAs have recoding signal
• Causes the ribosome to change its reading frame
• shift to –1 frame or to +1 frame
• common in viral mRNA’s
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Termination
• Stop codons (UAA, UGA, UAG)
– Selenocysteine in ~12 mammalian proteins
• Selenocysteine rare, contains selenium (the 21st amino
acid)
• Has its own tRNA - tRNASec, but lacks its own AAS
• seryl-tRNA synthetase attaches serine to 3' end of
tRNASec
• After attachment, serine is altered enzymatically
• encoded by the stop codon UGA
• UGA followed by folded region of the mRNA that binds
special EF
• EF recruits a tRNASec into the A site rather than
termination factor
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Termination
• Termination requires release factors
– Bacteria have 3
• RF1 recognizes UAA & UAG
• RF2 recognizes UGA & UAA
• RF3 merely increases activity of other factors
– Eukaryotes have 2 (eRF1 & eRF3)
• work together & recognize all of the stop codons
• Release factors superficially resemble tRNA
• Enter A site and interact directly with stop codon
• A tripeptide acts as anticodon
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Termination
• Termination requires release factors
– RF3 (or eRF3) carries a bound GTP that is
hydrolyzed later
– Then polypeptide is severed from its
attachment to tRNA
– both deacylated tRNA & the release factor
are then released
– then ribosome separates from mRNA &
dissociates
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Non-sense mutations
• Cause a variety of inherited diseases
• Partial polypeptides can result
• Many such mRNAs destroyed by
nonsense-mediated decay (NMD)
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Polyribosomes
• Also known as polysomes
– Greatly increases protein synthesis rate
– Polysomes free in cytosol synthesize soluble
proteins
– also seen on cytosolic surface of ER
• make membrane, secretory and/or organelle proteins
• Coupled transcription/translation
– Possible only in Prokaryotes
– In eukaryotes, mRNA must leave nucleus
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Copyright, ©, 2002, John Wiley & Sons, Inc.,
Karp/CELL & MOLECULAR BIOLOGY 3E
Figure 11.51a