Translation 翻译

• Ribosomes hold the mRNA and tRNAs together and connect the amino acids at the A site to the growing polypeptide.
Ribosome Structure
Structure of tRNA
CapCap-Dependent Initiation of Protein Synthesis in Eukaryotes
An initiation complex forms at the cap with the 40S ribosomal subunit and other translation initiation factors. The 40S complex then scans down the 5’ untranslated region to the first AUG codon. A GTP hydrolysis step by eIF5 triggers GDP binding of eIF2 and release of initiation proteins. The 60S subunit joins the complex and the 80S ribosome initiates translate the ORF.
Elongation
Ribosome selects aminoacylated tRNA eEF1α and GTP are bound to α aminoacylated tRNA Ribosome catalyzes formation of a peptide bond Translocation is dependent on eEF2 and GTP hydrolysis Many ribosomes may translate mRNAs simultaneously on the same strand.
•Small & large ribosomal subunits.
•A Binding site for the mRNA is present on small subunit. •Two binding sites (P & A) bind tRNAs on large subunit.
–P site – holds the tRNwenku.baidu.com carrying the growing polypeptide chain. –A site – holds the tRNA with the next AA to be added.
5’end (cap) independent initiation:
Poliovirus
• mRNAs of picornaviruses lack 5’ cap
• Ribosomes bind internally rather than at the mRNA 5’ end • 5’ end of poliovirus RNA promotes internal binding of 40S subunit at internal ribosome entry site (IRES) • In poliovirus infected cells eIF4G is cleaved, inactivating translation of cellular mRNAs •The initiation in the IRES does not depend on the presence of a cap structure, but requires C-terminal fragment of eIF4G to recruit the 40S subunit through interaction with eIF3.
Many RNA Viruses have capped genomic RNAs similar to eukaryotic host mRNAs
• Most eukaryotic mRNAs are capped at the 5’ end during nuclear processing. • The terminal 5’ phosphate is first removed by a 5’ triphosphatase. • Guanyltransferase transfers GMP from GTP to the 5’ end of the mRNA to add the GpppN cap structure. • The 5’ terminal inverted G residue is then modified by methylation. • Many RNA viruses replicate in the cytoplasm and must use a viral dependent capping mechanism supplied by the RNA-Dependent-RNA Polymerase. • The Cap structure, m7GpppN, is most common in viral and mammalian mRNAs.
5’ end (cap) dependent initiation:
• The first step is the recognition of the 5’ cap by eIF4F, which consists of three proteins, eIF4E, eIF4G and eIF4A. • Cap binding protein, eIF4E, binds to cap • The N-terminus of eIF4G binds eIF4E and the Cterminus binds eIF4A • The 40S subunit binds to eIF4G via eIF3
• Aligns each amino acid with the corresponding codon • 70-80 nt long • 3’ end has the 5’- CCA sequence to which aa are linked • The opposite end contains the anticodon loop • Contains modified bases
Three distinct stages of translation Initiation
•Rate limiting step •Requires hydrolysis of ATP and GTP •Results in formation of a complex containing the mRNA, the ribosome and the initiator Met-tRNA A. 5’ end (Cap) dependent initiation • The initiation complex binds to the 5’ cap structure and scans in a 5’ to 3’ direction until initiating AUG is encountered B. Internal ribosome entry • Initiation complex binds upstream of initiation codon
Structure of eukaryotic and prokaryotic mRNAs:
Model of eukaryotic ribosome
• rRNAs are believed to play a catalytic role in protein synthesis. • After removal of 95% of the ribosomal proteins, the 60S subunit can catalyze formation of peptide bonds. • Ribosomal proteins are now believed to help fold the rRNAs properly and to position the tRNAs.
Five different types of IRES sequences are found on viral RNAs: Type I-entero and rhinoviruses (poliovirus) Initiation codon is located past the 3’ end of the IRES 40S binds to IRES scans to AUG Type II-cardio and apthoviruses (EMCV) Initiation codon is at the 3’ end of the IRES 40S binds at or near AUG no scanning occurs Type III- hepatitis A virus initiation codon is located past the 3’ end of the IRES requires all of initiation proteins, including eIF4E Type IV- hepatitis C virus The 3’ end of the hepatitis C virus IRES extends beyond the AUG codon Type V-cricket paralysis virus IRES ends at the initiation codon, although it is not an AUG codon, no initiation factors are required initiation codon is placed at the A site instead of the P site
eEF2 GTP P A
eEF1α α GTP
Termination •Translation is terminated at one of three stop codons (UAA, UAG & UGA). • Termination codon at the A site is recognized by the release factor instead of a tRNA • The release factor binds the termination codon • The peptide chain is then released followed by dissociation of the tRNA and the ribosome
5’
Promoter/ Control Region
Exon 1 Int. 1
Exon 2 Int. 2 Exon 3
3’
Terminator Sequence
Exons
Transcription
5’
Exon 1 Int. 1
Exon 2 Int. 2 Exon 3
3’
Unprocessed RNA Transcript
Eukaryotic mRNA Transcripts are Processed
Cytoplasm
DNA
Transcription
RNA
RNA Processing
mRNA
G
AAAAAA
G
AAAAAA
Export
Nucleus
A “Simple” Eukaryotic Gene
Transcription 5’ UTR Start Site Introns 3’ UTR
Closed loop model:
• The 5’ end dependent initiation is stimulated by the poly(A) binding protein Pabp1p, which interacts with eIF4G • This interaction circularizes the mRNA and facilitates formation of the initiation complex • Mechanism to ensure that only intact mRNA is translated
1) Removal of Introns 2) Addition of a 5’ cap 3) Addition of a 3’ tail The mRNA then moves out of the nucleus and is translated in the cytoplasm.
Translational control
Processing of eukaryotic mRNA
5’ UTR Protein Coding Region 3’ UTR
G
5’ Cap
Exon 1 Exon 2 Exon 3
AAAAA
3’ Poly A Tail
• RNA processing achieves three things: