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![Reading a mRNA
[5’cap]CCGGACUACCUCUGGACCCCCTCCCCU
GUCCCCAACGCUGAGCCGAAACC AUG CAU
GGG CGC CUG AAG GUA AAG ACG UCG GCU
GAA GAG CAG GCA GAG GCC AAA AGG CUA
GAA CGA GAG AAG CUA AAG CUC UAC CAG
UGA GCCACUCAAGCUGUCUUCAAAAn 3’
• Ribosome scans from 5’ end of mRNA (cap)
• Translation starts at first AUG, continues in frame, i.e. with
immediately succeeding triplet codon (CAU) and so on…..
• Translation stops at first in frame termination codon](https://image.slidesharecdn.com/proteintrans-250624070516-be87acf8/75/protein-translation-and-post-translational-modification-10-2048.jpg)
![Translation:
mRNA
Initiation
Factors:
eIF-2
eIF4E, G
U G C G G A U G C G A U G G A A A U U C
a) Initiation
U A C
Met
Small Ribosomal
Subunit
40S
Step 2: assembly of pre-initiation complex
GTP
[5’cap]
• Only initiator Met-tRNA can bind to 40S subunit alone
• 40S subunit is primarily involved in tRNA and mRNA recognition
3’](https://image.slidesharecdn.com/proteintrans-250624070516-be87acf8/75/protein-translation-and-post-translational-modification-16-2048.jpg)
![Translation:
mRNA
U G C G G A U G C G A U G G A A A U U C
U A C
Met
a) Initiation
40S
Step 3: binding of mRNA to preinitiation complex;
initiator Met binding sets the frame of the translation
eIF-2 GTP
[5’cap]
• eIF4E and G bind to cap and are recognised by 40S/Met-
tRNA/eIF2
eIF4E, G
3’](https://image.slidesharecdn.com/proteintrans-250624070516-be87acf8/75/protein-translation-and-post-translational-modification-17-2048.jpg)
protein translation and post translational modification
- 1. Protein translation andpost translational modification Professor Tony Magee Course code (MBBS) : Nucleic acids 5 Course code (BMS) : NAGE-L6
- 2. Learning Objectives At theend of this lecture the students will be able to: • Outline the mechanisms by which ribosomes can translate a mRNA sequence into a protein sequence • Describe the role of aminoacyl tRNAs in ensuring the fidelity of the genetic code • State how a ribosome recognises the start and end of a sequence to be translated • Explain why some antibiotics inhibit protein synthesis in prokaryotes but not eukaryotes • Identify the features of a newly-synthesised protein that are required for it to enter the secretory pathway • Give examples of the ways in which newly-synthesised proteins can be post-translationally modified e.g. insulin
- 3.
- 4.
- 5. • There isa linear relationship between the information encoded within DNA and the proteins which are synthesised using that information • Eukaryotic DNA contains introns (non-coding) and exons (coding) • Transcription of DNA -> pre-mRNA -> SPLICING (removal of introns) -> mRNA Three nucleotides encode one amino acid Exon DNA 300nt Protein 100 amino acids A group of three nucleotides is called a codon via mRNA
- 6. 5’cap 5’UTR codingregion 3’UTR polyA Structure of a typical mRNA 7MeG AAAAAn 5’ “cap” (7-Methyl Guanosine) – entry site for ribosome UTR – untranslated region polyA – protects mRNA
- 7. U C AG U C A G U C A G U C A G U C A G U C A G UUU UUC UUA UUG CU A C A C A CA A CG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG UCU UCC UCA UCG UAU UAC UAA UAG UGU UGC UGA UGG C C C C CU CC CA CG G G G G CU CC CA CG CAU CAC CAA CAG CGU CGC CGA CGG A A A G U A C A A A A A A A A G U A C A G G G G GAU GAC GAA GAG G U G C G A G G G G G G The genetic code (U=RNA, T=DNA) “wobble” position first base second base
- 8. U C AG U C A G U C A G U C A G U C A G U C A G Phe Phe Leu Leu Leu Leu Leu Leu Ile Ile Ile Met Val Val Val Val Ser Ser Ser Ser Pro Pro Pro Pro Thr Thr Thr Thr Ala Ala Ala Ala Tyr Tyr STOP STOP His His Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu Cys Cys STOP Trp Arg Arg Arg Arg Ser Ser Arg Arg Gly Gly Gly Gly Thr Thr Thr Thr There are 64 codons for 20 amino acids “wobble” position first base second base
- 9. U C AG U C A G U C A G U C A G U C A G U C A G Phe Phe Leu Leu Leu Leu Leu Leu Ile Ile Ile Met Val Val Val Val Ser Ser Ser Ser Pro Pro Pro Pro Thr Thr Thr Thr Ala Ala Ala Ala Tyr Tyr STOP STOP His His Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu Cys Cys STOP Trp Arg Arg Arg Arg Ser Ser Arg Arg Gly Gly Gly Gly Start (Met) = AUG Stop = UAA UAG UGA Rare amino acids have fewer codons e.g. Met, Trp
- 10. Reading a mRNA [5’cap]CCGGACUACCUCUGGACCCCCTCCCCU GUCCCCAACGCUGAGCCGAAACCAUG CAU GGG CGC CUG AAG GUA AAG ACG UCG GCU GAA GAG CAG GCA GAG GCC AAA AGG CUA GAA CGA GAG AAG CUA AAG CUC UAC CAG UGA GCCACUCAAGCUGUCUUCAAAAn 3’ • Ribosome scans from 5’ end of mRNA (cap) • Translation starts at first AUG, continues in frame, i.e. with immediately succeeding triplet codon (CAU) and so on….. • Translation stops at first in frame termination codon
- 11. mRNA is transportedout of the nucleus and translated in the 5’->3’ direction into protein (N->C direction) in the cytoplasm and on the rough endoplasmic reticulum The machinery for translating mRNA is the ribosome, a large 2 subunit complex of proteins and ribosomal RNA (rRNA) Messenger RNA (mRNA) is transcribed from DNA (pre-mRNA) and processed (spliced) in the nucleus
- 12. S = Svedbergunit of sedimentation in a centrifuge (size) Nobel Prize Chemistry 2009; Yonath, Steitz and Ramakrishnan
- 13. 3´ end 5´ end Anticodon AUG UAC Codon(wobble) Amino acid transfer RNA: (>1 for each aa) the transporter of amino acids to the ribosome antiparallel binding - like DNA 5’ 3’ (Met) mRNA
- 14. Aminoacyl tRNA synthetases AminoAcid ATP PPi E E -AMP-Amino Acid AMP E Amino Acid (Adenylated aa) • one for each aa • important role in fidelity of translation (selectivity for correct aa; hydrolysis of incorrect aa-tRNA) Mutated in cancers, neuropathies, autoimmunity, metabolic disease
- 15. Translation: a) Initiation Step1: dissociation of ribosome subunits (40S + 60S) Step 2: assembly of preinitiation complex containing Met-tRNA + eIFs + 40S subunit Step 3: binding of mRNA to preinitiation complex Step 4: binding of 60S subunit (simplified version; for details see Alberts et al., Molecular Biology of the Cell, 4th ed.)
- 16. Translation: mRNA Initiation Factors: eIF-2 eIF4E, G U GC G G A U G C G A U G G A A A U U C a) Initiation U A C Met Small Ribosomal Subunit 40S Step 2: assembly of pre-initiation complex GTP [5’cap] • Only initiator Met-tRNA can bind to 40S subunit alone • 40S subunit is primarily involved in tRNA and mRNA recognition 3’
- 17. Translation: mRNA U G CG G A U G C G A U G G A A A U U C U A C Met a) Initiation 40S Step 3: binding of mRNA to preinitiation complex; initiator Met binding sets the frame of the translation eIF-2 GTP [5’cap] • eIF4E and G bind to cap and are recognised by 40S/Met- tRNA/eIF2 eIF4E, G 3’
- 18. eIF-2 GTP Translation: mRNA U GC G G A U G C G A U G G A A A U U C U A C Met GTP GDP + Pi (ensures correct base pairing) a) Initiation Step 4: binding of 60S subunit 40S 60S 3’ GDP
- 19. Translation: mRNA U G CG G A U G C G A U G G A A A U U C U A C Met GTP GDP + Pi a) Initiation Step 4: binding of 60S subunit 40S 60S eIF-2 GDP 3’
- 20. Translation: b) Elongation Step1: binding of new tRNA carrying second amino acid to A (amino acyl) site Step 2: catalysis of peptide bond between the two amino acids by peptidyl transferase Step 3: translocation of tRNA to P (peptidyl) site and dissociation of first tRNA
- 21. Translation: mRNA U A C Met b)Elongation U G C G G A U G C G A U G G A A A U U C P site A site G C U Arg Step 1: binding of new tRNA to immediately adjacent A site in frame with initiator Met 3’
- 22. Translation:b) Elongation Step 2:catalysis of peptide bond between the two amino acids by peptidyl transferase on 60S subunit mRNA U A C Met U G C G G A U G C G A U G G A A A U U C P site A site G C U Arg PT 3’
- 23. Translation: mRNA b) Elongation U GC G G A U G C G A U G G A A A U U C P site A site Met U A C G C U Arg Step 2 continued: 3’
- 24. Translation: mRNA b) Elongation P siteA site Met U A C G C U Arg U G C G G A U G C G A U G G A A A U U C Step 3: translocation of peptidyl tRNA to P site 3’ • Elongation Factors (EFs) are proteins that promote movement of ribosome along mRNA using GTP
- 25. Translation: mRNA b) Elongation P siteA site U G C G G A U G C G A U G G A A A U U C Met G C U Arg U A C Step 3 (continued): dissociation of first tRNA 3’
- 26. Translation: mRNA b) Elongation P siteA site U G C G G A U G C G A U G G A A A U U C Met G C U Arg A C C Trp New cycle with new step 1 3’
- 27. Translation: mRNA b) Elongation P siteA site U G C G G A U G C G A U G G A A A U U C A C C Met Arg Trp G C U New step 2 3’
- 28. Translation: mRNA b) Elongation P siteA site A C C Met Arg Trp G C U U G C G G A U G C G A U G G A A A U U C New step 3 3’
- 29. Translation: mRNA b) Elongation P siteA site A C C Met Arg Trp G C U U G C G G A U G C G A U G G A A A U U C Etc., etc. EFs use the energy of GTP to enhance the efficiency and accuracy of translation by providing “pauses” (e.g. GTP hydrolysis) that allow incorrect base pairs to dissociate 3’
- 30. Translation: c) Termination Step1: recognition of stop codon Step 2: release of peptide chain Step 3: dissociation of release factors and ribosomes
- 31. Translation: mRNA c) Termination C GU G A A U C A G U A A U U C G A A U G U C Gln Release factors (proteins, not tRNAs) bind to empty A site Step 1: recognition of stop codon RF 3’ Out of frame UGA
- 32. Translation: mRNA c) Termination C GU G A A U C A G U A A U U C G A A U RF Gln G U C Step 2: release of peptide chain Peptidyl transferase catalyses transfer of the completed protein chain to water and releases it from the ribosome 3’
- 33. Translation: mRNA c) Termination C GU G A A U C A G U A A U U C G A A U Gln G U C RF 40S 60S 3’
- 34.
- 35. Polyribosomes Ribosomes do notwork singly on a mRNA but in multiple copies on the mRNA – a polyribosome – like a string of beads http://www.sumanasinc.com/webcontent/animations/content/ polyribosomes.html
- 36. Translation speed • Translationspeed of each ribosome = 15 amino acids/sec • Multiple ribosomes processing simultaneously a 300 a.a. long protein, i.e. one ribosome every 30a.a. of synthesized protein - the number of protein molecules produced in 1 min is ~4000
- 37. Many antibiotics inhibitprotein synthesis in prokaryotes • Translational machinery is complex, easily disrupted – common target for antibiotics • Antibiotics selectively inhibit prokaryotes • Antibiotics are natural products of bacteria or fungi to give them a selective advantage over other microbes • Antibiotics exploit differences between prokaryotic and eukaryotic ribosomes and translation factors
- 38. Antibiotics inhibiting proteinsynthesis • Streptomycin Inhibits initiation • Tetracycline Inhibits aa-tRNA binding • Erythromycin Inhibits translocation • Chloramphenicol Inhibits peptidyl transferase • Puromycin Terminates elongation prematurely
- 39. Plasma membrane Lysosome Golgi Apparatus Ribosome RoughEndoplasmic Reticulum Vacuole Nucleus Peroxisome Nucleolus Mitochondrion Smooth Endoplasmic Reticulum Cytosol INTRACELLULAR COMPARTMENTS Protein synthesis takes place in the cytoplasm Most cellular compartments are bounded by a membrane so the cell needs a mechanism to transfer proteins across membranes
- 40. Secretory and transmembraneproteins are synthesised in the Rough Endoplasmic Reticulum Same ribosomes on RER as for cytoplasmic proteins
- 41. Protein synthesis on RoughEndoplasmic Reticulum (RER): secreted and transmembrane proteins First 20-24 amino acids = “signal sequence” (hydrophobic amino acids, e.g. Leu, Ile, Phe, Trp, Tyr, Ala) 40S 60S mRNA
- 42. Protein synthesis onRER RER SRP Receptor SRP Step 1: recognition of signal sequence by a protein-RNA complex “Signal-Recognition Particle” (SRP), halting translation mRNA 40S 60S cytoplasm lumen
- 43. Protein synthesis onRER RER SRP Receptor Step 2: binding of SRP to a receptor at the RER surface, translation resumes SRP 40S 60S
- 44. Protein synthesis onRER RER Protein channel Step 3: translocation into the lumen of RER 40S 60S SRP
- 45. Protein synthesis onRER RER lumen Cytoplasm 40S 60S Transmembrane proteins have an extra hydrophobic sequence holding them in the membrane Secreted protein
- 46. Protein synthesis onRER Step 4: cleavage of signal sequence by signal peptidase (co- translational) and folding
- 47. Post-translational modification - Aftersynthesis most proteins are modified further before they are fully functional - Only 20 amino acids – cell uses post-translational modifications (over 200) to increase diversity, including: • Disulphide bond formation (e.g. insulin) • Proteolytic cleavage (e.g. insulin -> A and B chains) • Addition of carbohydrate (Glycosylation) • Addition of phosphate (Phosphorylation) • Addition of lipid groups (Prenylation, Acylation) • Hydroxylation (e.g. Collagen; Leitinger lecture)
- 48. Insulin biosynthesis in pancreaticβ cells Insulin undergoes extensive post- translational modification along the production pathway, including disulphide bond formation in the ER and proteolytic cleavage in the secretory vesicle to produce active insulin
- 49. Glycosylation in theRER and Golgi complex Golgi RER Pre-assembled carbohydrate chains N-linked to Asn of (AsnXSer/Thr) “sequon” PM Initial N-glycosylation Glycan processing ( ) Carbohydrate chains processed by trimming followed by extension Transport vesicles Glycan on lipid-linked precursor