Definitions tran·scrip·tion (noun): the act of making an exact copy of a document. –Example: the...
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Transcript of Definitions tran·scrip·tion (noun): the act of making an exact copy of a document. –Example: the...
Definitions• tran·scrip·tion (noun): the act of making an exact copy
of a document.
– Example: the very old method for making a copy of a book by hand.
• trans·la·tion (noun): the rendering of the meaning of something into a different language.
– Example: translating Leo Tolstoy’s novel “War and Peace” from Russian (the original) into English.
Translation• The synthesis of a protein polymer from a RNA template
– The ribosome translates the chemical language of nucleic acids to amino acids– Provides a control point for regulation of gene expression– Amplification step (can make many protein copies)
Translation• There must be a nucleic acid code for amino acid sequences
– 4 different nucleic acid bases, 20 different amino acids– PLUS, need information about where to START and where to STOP translating– Possible CODON sizes:
• 1 base 41 = 4 not big enough
• 2 bases 42 = 16 not big enough
• 3 bases 43 = 64 THIS WOULD WORK
• The code could be overlapping or NONOVERLAPPING• Nonoverlapping is less sensitive to mutation
Translation• Codons are nonoverlapping 3 nucleotide units
– START = AUG (Methionine)– STOP = UGA, UAG, UAA (does NOT also encode an amino acid)
– 61 of 64 codons are left for amino acids• There are only 20 amino acids• The code is “degenerate” with several codons per amino acid
– CUN = Leucine– UCN = Serine– CCN = Proline– ACN = Threonine (ACA, ACG, ACC, ACU)
(Where N = A, G, C or U)
**Note, much of the degeneracy is in the 3rd position of the codon**
Transfer RNAs (tRNA)• Bridge between nucleic acid and
amino acid languages
– 73 - 93 nts long– Several modified bases (e.g.
pseudouridine, etc)– Complementary regions base pair
to form cloverleaf-like structure
• Packs further to look like: • Amino acid attached to 3’-OH
via ester linkage• Anticodon loop basepairs with
mRNA codon
Transfer RNAs (tRNA)• Degeneracy of code
– Lots of tRNA genes– 1 tRNA can recognize > 1 codon
• Strict base pair rules for codon position 1 and 2
• “wobble” in position 3– Non Watson-Crick pairing
e.g. G:U pairing
Charging tRNAs • Accuracy for protein synthesis is
primarily from the accuracy of attaching the correct amino acid to the correct tRNA– 20 Aminoacyl tRNA synthetase enzymes– 1 enzyme for each amino acid– 1 enzyme can recognize >1 tRNA
• Specificity from interactions with acceptor and anticodon arms of tRNA
Charging tRNAs and proofreading
• tRNA synthetase enzymes can proofread– Can hydrolyze wrong amino acid from tRNA
The ribosome • Large RNA-Protein complex
• Large ribosomal subunit (60S)• Small ribosomal subunit (40S)
• Steps in translation:
INITIATION– Bind mRNA, find start
ELONGATION– Find next amino acid, add it
TERMINATION– Recognize stop, and release
Translation mechanism: bacteria • INITIATION
– Small subunit binds “Shine-Dalgarno” sequence in mRNA
5’-AGGAGG-3’
DNA: 5’-…TATAAT n n n n A n n n n AGGAGG n n n n n ATG…-3’-10 +1
mRNA: 5’-A n n n n AGGAGG n n n n n AUG…-3’
Translation mechanism: bacteria • INITIATION
– Small subunit binds Shine-Dalgarno sequence in mRNA to locate AUG
– INITIATION FACTORS
1) IF1, IF2, IF3
• IF2 binds GTP
Translation mechanism: bacteria • INITIATION
– Small subunit binds Shine-Dalgarno sequence in mRNA to locate AUG
– INITIATION FACTORS
1) IF1, IF2, IF3
• IF2 binds GTP
2) IF2 binds initiating tRNA-Met
Translation mechanism: bacteria • INITIATION
– Small subunit binds Shine-Dalgarno sequence in mRNA to locate AUG
– INITIATION FACTORS
1) IF1, IF2, IF3– IF2 binds GTP
2) IF2 binds initiating tRNA-Met
3) Recruit large subunit, release IF1, IF3
– If codon-anticodon interaction is correct, IF2 hydrolyzes GTP and leaves
Translation mechanism: eukaryotes • INITIATION
– A pre-formed eIF1, 2, 3 + Small subunit complex binds 5’-CAP region– eIF4 and other factors are involved in sensing additional features of mRNA
• 5’-CAP structure• 3’-end polyA tail
– Complex SCANS 5’--> 3’ for the consensus sequence 5’-CCACCAUG-3’
start codon
Translation mechanism: bacteria • ELONGATION
– After large subunit bound, 3 sites are present in ribosome• Aminoacyl (A) site• Peptidyl (P) site• Exit (E) site
– tRNA-MET in P site
1) EF-Tu + GTP + Phe-tRNA bind in A site
– If codon-anticodon interaction is proper, hydrolyze GTP --> GDP, release EF-Tu
Translation mechanism: bacteria • ELONGATION
– If codon-anticodon interaction is proper, hydrolyze GTP --> GDP, release EF-Tu
2) Peptidyl transferase enzyme catalyzes bond formation• large subunit of
Ribosome RNA is a Ribozyme!
– Met is now attached at the “A” site: Met-Phe-tRNA
Translation mechanism: bacteria • ELONGATION
– Whole ribosome must be translocated 3 nts downstream on mRNA
3) EF-G hydrolyzes GTP for translocation reaction
– tRNA in P site is now in E– Met-Phe-tRNA is now in P
– Repeat ELONGATION cycle until a stop codon is reached
Translation mechanism: bacteria • ELONGATION
– Repeat ELONGATION cycle until a stop codon is reached
– EF-Tu + GTP + Ser-tRNA --> EF-Tu + GDP
(note exit of tRNA from E site)
– Peptidyl transferase activity would yield Met-Phe-Ser-tRNA in A site
– And so on…
Translation mechanism: bacteria • TERMINATION
– Stop codons are not recognized by any wildtype tRNAs
– Three Release Factor proteins• RF1• RF2• RF3
– Enter A site and trigger hydrolysis of Met-Phe-Ser from tRNA
– Large and small subunits dissociate from mRNA template
U A A
Polyribosomes • mRNAs can be translated by multiple ribosomes at same time• Amplification step in gene expression
Coupled TXN & TLN: bacteria• A gene can be transcribed and
translation of it can start before TXN is finished
Frameshift mutations• Consider the following mRNA sequence
• Frameshift mutations insert or delete one or more bases into an Open Reading Frame (ORF)– Insert one base
– Delete one base
5’-GCCUCAGGAACCACC AUG CUA GCU UGC UGAAAUAAAAAAAAAAA-3’TLN: M L A C *(stop)
5’-GCCUCAGGAACCACC AUG CUC AGC UUG CUG AAA UAA AAAAAAAAATLN: M L S L L K *(stop)
5’-GCCUCAGGAACCACC AUG UAG CUUGCUGAAAUAAAAAAAAAAA-3’TLN: M *(stop)
Nonsense mutations & nonsense mediated decay• Consider the following mRNA sequence
• Nonsense mutations change an amino acid codon to a stop codon
• If this mutation is in any exon other than the last one,• Nonsense mediated decay (NMD) will block translation of it
5’-GCCUCAGGAACCACC AUG CUA UGG UGC UGAAAUAAAAAAAAAAA-3’TLN: M L W C *(stop)
5’-GCCUCAGGAACCACC AUG CUA UGA UGC UGAAAUAAAAAAAAAAA-3’TLN: M L *(stop)
Nonsense mutations & nonsense mediated decay• Exon-Junction Complex (EJC) proteins are deposited on
transcripts ~20 nts upstream of new Exon-Exon junctions• Ribosome knocks them off during TLN• If ribosome doesn’t knock them off, transcript is destroyed
– Wildtype mRNA:
– Nonsense mutation mRNA:
– EJC that is not removed generates a signal targeting mRNA for destruction
XEJC EJC EJC
XX EJC EJC EJC