Transcript Translation is the process where mRNA codons are used to produce

Translation is the process where mRNA codons are used
to produce protein 11/23
•
•
•
•
•
•
•
•
•
•
•
What are the basics of translation?
What are the 64 codons? Why only 61 tRNAs?
How do eukaryotic and prokaryotic mRNAs differ?
How does eukaryotic and prokaryotic translation differ?
What are ribosomes composed of?
What are the 4 ribosomal binding sites?
How is tRNA “charged” with an amino acid?
How is translation “initiated”
What sets the “reading frame” of the codons in mRNA?
How does peptide chain “elongation” occur?
What “terminates” mRNA translation?
Transcription Review: how did we make mRNA?
The DNA duplex is unwound by RNAP to create access to the
template strand for transcription into a complementary RNA
sequence. NTPs are ONLY added (elongation) to the RNA 3’ end
Translation is the process whereby the mRNA sequence is
converted into a protein sequence.
• Eukaryotic and Prokaryotic requirements are very similar!
– 1) Ribosome: Large subunit and Small subunit (Svedberg Units)
– 2) tRNAs are required to carry the 20 different amino acids
– 3) mRNA must have a start sequence (AUG) and a stop codon
• mRNA characteristics:
– Euk: mRNA is modified with a 5’-CH3 cap and 3’polyA-tail
– Prok: mRNA has a special ribosomal binding site just upstream of AUG
• Shine Delgano Sequence critical for initiation to occur.
• Three Main Processes to Translation of mRNA:
– 1) Initiation: mRNA Bound by ribosome, then tRNA binds AUG
– 2) Elongation: amino acids brought to new peptide by tRNAs
– 3) Termination: peptide and ribosome released from mRNA
The first 5’A-U-G-3’ encountered after the 5’ end of the mRNA is the
start “codon”. Each triplet of bases after this is a specific “codon”.
There are 20 standard amino acids
There are 4X4X4=64 codons
Base triplet is a codon
61 codons are for amino acids
3 codons terminate translation
AUG codes for methionine
AUG Codes for “Start”
Termination occurs with:
UAA UAG and UGA
FrameShift Mutations change
protein sequence by changing
codon triplet identities on
mRNA template
CUGAUGA Add “A”
 CAUGAUGA  new protein
Position if 3rd nucleotide is the
most variable “Wobble”
Ribosomes are massive assemblies of proteins and pieces
of rRNA that form discrete “small” and “large” subunits.
Prokaryotes:50S+30S=70S Eukaryotes:60S+40S=80S
Ribosomes work free in the cytosol or when membrane bound
i.e. Rough Endoplasmic Reticulum (RER)
Small ribosomal subunit bind mRNA first, then Large subunit
Complex has four RNA-lined binding sites
1) mRNA-Site
2) A-Site (aminoacyl-tRNA)
3) P-Site (peptidyl-tRNA
4) E-Site empty tRNA exits from here!
We call a ribosome a “ribozyme” because it has enzyme-like activity but it
is made of RNA at its active site, not protein!
The Proteins associated with it help regulate its shape and activity but are
not entirely critical for its activity
The four binding sites on the ribosome are located deep inside the
complex and the sites straddle the small and large subunits.
Ribosomes consists of 2/3 rRNA and 1/3 Protein by weight.
tRNAs must be “charged” or “activated” by having their specific
amino acid added to the hydroxyl located on their 3’ end by a
special enzyme called aminoacyl-tRNA synthetase before
translation can progress.
Some amino acids have several different codons
There are 20 different amino acids
Each amino acid type has a specific aminoacyl-tRNA
synthetase that recognizes all codons associate with that
specific amino acid
There are 20 different kinds of aminoacyl-tRNA synthetase
AA+ATP+tRNAaminoacyl-tRNA(charged)+AMP+ 2 Pi
“Charging costs two Pi-Pi bonds! Expensive
tRNA Anticodon binds complementary mRNA Codons
mRNA ‘5-AUG-3’ binds ‘3-UAC-5’ of tRNA
RNA=RNA binding is not as stable as DNA Double helix
“Wobble” refers to the fact that the bases in the codon
(especially the third) can be variable.
How is the translation of mRNA to protein initiated?
Four Steps to Initiation:
• 1) Small ribosomal subunit binds 5’ end of mRNA
• 2) met-tRNA binds ribosome and crawls along mRNA until an AUG is encountered
• 3) Small subunit binds AUG and then attracts the large ribosomal subunit
• 4) Large subunit binds to small unit/mRNA to complete the ribosomal complex
• Initiation is now completed!
•
Note:
– The first AUG encountered by the ribosome sets the “Reading Frame” for all
base-triplets (codons) that come after it, mRNA is read 3 bases at a time.
– A single mRNA can have several ribosomes on it at one time
– In prokaryotes a Shine-Delgano Sequence of –AGGA- must precede the AUG
for initiation (not required in eukaryotes)
• Prokaryotic Shine-Delgano sequence on mRNA ‘5-AGGA-3’ binds ‘3-UCCA5’ on the complementary rRNA stabilizing initiation complex.
– In prokaryotes the first amino acid is always formyl-methionine
• fMet => Methionine with a formyl group on its amino end to make it nonreactive while being created
Elongation: additional charged tRNAs bring additional amino acids to the
open A-site on the ribosome. The new aminoacyl-tRNA accepts the old
peptide chain. It becomes the P-site and the old tRNA shifts into the E-site.
Peptide elongation process uses a “ribozyme” called peptidyltransferase.
Termination of translation
occurs when a stop codon
UAA, UAG or UGA is
exposed at the A-site.
Stop codon causes a
release factor (protein) to
bind this open site causing
the nascent peptide to be
released from the 3’-end of
tRNA at the P-site.
After release, the ribosome
and mRNA also dissociate.
Start Codon: AUG Termination Codons: UAC UAG and AGA
1) Given this mRNA how many amino acids long is the protein?
5’UUCGAUG-GCC-UCU-UGC-AUG-GCG-UAG-UUU-AG…3’
1
5
10
30
2) If Base #5 (A) is removed a new reading frame is established, where does
synthesis start?
5’UUCGUGGCCUCUUGC-AUG-GCG-UAG-UUU-AG….3’
1
5
10
go
29
3) If an extra base (G) is ADDED immediately after #7 the total mRNA
becomes 31 bases, how many amino acids are produced?
5’UUCGAUG-GGC-CUC-UUG-CAU-GGC-GUA-GUU-UGC...3’
1 5go
10
31