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**

Reading the codon table

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

Transfer RNAs (tRNA)• Examples of tRNAs tolerating G:U pairs in codon position 3

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’


– Small subunit binds Shine-Dalgarno sequence in mRNA to locate AUG

– INITIATION FACTORS

1) IF1, IF2, IF3

• IF2 binds GTP




1) IF1, IF2, IF3

• IF2 binds GTP

2) IF2 binds initiating tRNA-Met




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


– 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


– 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


– 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

definitions tran·scrip·tion (noun): the act of making an exact copy of a document. –example: the...

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