Translation

Chapter 27

Central Dogma• Genetic code contained w/in 4 deoxy-

nucleotide bases of DNA

• 1 gene 1 polypeptide

• DNA is template for codes, but not direct template

– DNA transcr’d mRNA

–mRNA is direct template for polypeptides

• Translation = cell uses mRNA to construct polypeptides

The Genetic Code• Discovered in 1960’s (27-7)

• Same code for almost all prokaryotes, eukaryotes

• Codon = 3 nucleotide bases of mRNA that code for 1 aa

– REMEMBER: this info was originally “held” as deoxynucleotides w/in gene of DNA

• Most aa’s have >1 codon

– Only aa’s w/ single 3-base codon = met, trp

– “Wobble” @ 3rd nucleotide

Fig.27-7

The Genetic Code – cont’d• AUG = initiation codon

– Codes for met

• UAA, UAG, UGA = stop codons

– Code for termination of polypeptide synth

• No spacing between codons

– Reading frame = 3 nucleotides

– If insertion or deletion frame shift (27-3)

• May improper aa’s inc’d into polypeptide

Ribonucleic Acids• Messenger RNA (mRNA) – described in

transcr’n (Chpt. 26)

– Size varies according to gene size

• So polypeptide size

– Contains genetic code w/ 3 nucleotides/aa

• Ribosomal RNA (rRNA) – RNA that helps make up the ribosome

• Transfer RNA (tRNA) – carries aa to proper site on ribosome

The Ribosome

• Complex of enz’s, rRNA’s, aa’s to accomplish translation

• Made up of subunits

• Different sizes rRNA, prot’s, subunits (27-11)

– Bacterial 70S = 50S + 30S subunits

– Eukaryotic 80S = 60S + 40S subunits

• S = Svedberg unit = size unit based on centrifugation properties

Fig.27-11

Fig.27-11

The Ribosome – cont’d

• rRNA’s have complicated 2o structures (27-12)

– Needed to position mRNA/tRNA

– May act like enzymes – assist in catalyzing form’n of peptide bonds

Transfer RNA (27-15)

• Interacts w/ both m and rRNA’s as well as aa’s

• At least 1 tRNA for each aa

• 1 region of tRNA becomes covalently linked to its aa

– “Amino acid arm”

Transfer RNA – cont’d• Covalent binding of aa to tRNA by tRNA

synthetase

– 1 synthetase enz for each diff tRNA

– Forms high-energy intermediate between tRNA + aa

• Anhydride link w/ AMP (27-16)

• “Activated”

• This intermediate links to aa arm

– Forms high energy ester linkage

– Energy held in these high potential energy bonds is used in peptide bond form’n

Fig.27-16

Fig.27-16

Transfer RNA – cont’d• tRNA synthetase has proofreading

ability

–Where proper aa “knows” to be att’d to correct tRNA

• 1 region of tRNA contains “anticodon”

– “Anticodon arm”

– Opposite Amino acid arm

– Has seq of 3 nucleotide bases which interact (base-pair) w/ codon of mRNA (27-14)

Fig.27-14

Transfer RNA – cont’d

• Other arms differ slightly in shape, nucleotides

– Help synthetases distinguish proper tRNAs (27-18)

• Also has sites for attachment to 70 or 80S rRNA and mRNA

“Ingredients” for Translation

• mRNA

– Contains genetic code for proper aa sequence to synthesize polypeptide

• tRNA attached to aa (through high energy bond)(27-17)

– This complexes w/ mRNA @ codon for aa

– First base of mRNA codon base-pairs w/ 3rd base of anticodon on tRNA (27-8)

Fig.27-17

Fig.27-8

“Ingredients” for Translation – cont’d

• rRNA assoc’d w/ proteins ribosome

– Ribosome draws all structures together properly to facilitate translation

• Note: translation has initiation, elongation, termination steps. Book uses E. coli system as model

Initiation of Translation

• Begins @ amino terminus of new polypeptide

• New polypeptide always begins w/ met

– BUT altered met: N-formylmethionine (p.1044)

– fMet has particular codon that specifies it

• AUG

• Called initiation codon

Initiation – cont’d• 30S subunit of ribosome binds IF-3 and IF-1

(27-22)

– IF-3 (or 1) = Initiation Factor 3 (or 1) (proteins) (Table 27-9)

– IF-3 prevents early binding of ribosomal subunits

– IF-1 prevents improper binds of tRNA to wrong site

• mRNA binds 30S-IF3-IF1 complex

– Binding such that initiation codon of mRNA is at specific site on subunit

• Called P site (for “Peptidyl” site)

• Lies next to A site (for “Aminoacyl” site)

Fig.27-22

Initiation – cont’d– Shine-Dalgarno sequence helps w/ proper

placement of mRNA into P site (27-23)

• Specific seq along mRNA @ partic site relative to intitiation codon

• Recognized by rRNA of 30S ribosome

• Helps “line up” mRNA initiation codon @ P site on 30S subunit

Fig.27-23

Initiation – cont’d• tRNA enters the structure

–Must be tRNA that carries fMet

– tRNA-fMet complex must be associated w/ IF-2

– IF-2 must be bound to GTP

• Get 30S-IF3-IF1 complexed with mRNA (w/ initiation codon @ P site), with anticodon region of tRNA-fMet-IF2-GTP base-paired to initiation codon

Fig.27-22

Initiation – cont’d• 50S subunit enters completed

initiation complex

– GTP cleaved GDP + Pi

– IF-1, -2, -3 disassociate

– Now have 70S ribosome

– Now P and A sites have completed conform’ns

– Now 3rd site (E or exit site) is formed next to P site

Fig.27-22

Initiation – cont’d

• 3 recognition sites hold the ribosome together

– Shine-Dalgarno seq holds ribosome to mRNA

– Codon-anticodon holds mRNA to tRNA

– P site conform’n holds ribosome to tRNA

Elongation – cont’d• A site on ribosome is empty

– mRNA w/ codon for next aa is @ A site

• tRNA bound to aa, w/ anticodon that matches codon for next aa is prepared (27-25)

– Binds EF-Tu (“Elongation Factor Tu”, a protein), which is bound to GTP

Complex of tRNA-aa-EF Tu-GTP

• Complex approaches empty A site on 70S ribosome

– “Placed” by base-pairing of codon/anticodon

Fig.25-25

Elongation – cont’d

• EF Tu-GTP cleaved from tRNA and GTP hydrolyzed EF Tu-GDP + Pi

– Allows time for proofreading of base pairing of codon/anticodon

– GDP cleaved

– EF Tu-GTP regenerated

1st aa (fMet) and aa2 in P and A sites, respectively

– Now in proper position to peptide bond

Elongation – cont’d

-NH2 of aa2 attacks carbonyl of fMet (27-26)

aa2 tRNA now has dipeptide attached

1st tRNA now empty (uncharged w/ aa)

– REMEMBER: aa had been activated by ester bond form’n w/ tRNA

– rRNA of 50S subunit may participate in peptide bonding by catalyzing rxn

Fig.27-26

Elongation – cont’d

• Ribosome moves 5’ 3’ along mRNA by distance of 1 codon (27-27)

– Called “translocation”

– Due to conform’l change of ribosome

– Requires EF-G att’d to GTP

• EF-G = translocase

• GTP cleaved from translocase and

• Also, GTP hydrolyzed GDP + Pi

Fig.27-27

Elongation – cont’d Empty tRNA @ E site, tRNA-aa2-fMet

@ P site, A site empty

–mRNA w/ codon for next aa is @ A site

– Empty tRNA is expelled from ribosome

– Next tRNA can now position next aa for form’n of another peptide bond

• Note: polypeptide is always attached to tRNA of last aa added @ ribosome

Termination of Translation

• Signaled by 1 of 3 stop codons (UAA, UAG, UGA) on mRNA (27-28)

–When stop codon is @ A site

– No tRNA recognizes these codons

– These codons ARE recognized by Release Factors (RF1, RF2, or RF3) – proteins

• One of these binds mRNA @ stop codon

– RF3 may stimulate release of polypeptide

Termination – cont’d

• tRNA w/ polypeptide chain att’d now “stalled” @ P site

– Ester bond between tRNA and peptide hydrolyzed

– Also interactions between tRNA and ribosome weaken

– Also interactions between 30S, 50S subunits weaken

– Ribosome dissociates

Fig.27-28

Polysomes

• >1 Ribosome at a time translates a single mRNA (27-29)

– Efficient use of single mRNA strand

• In bacteria, mRNA transcript is translated before transcr’n complete (27-30)

– REMEMBER: no nucleus in prokaryotes, so repl’n, transcr’n, transl’n all occur in cytoplasm

• Not true of eukaryotes

Fig.27-29

Fig.27-30

New Proteins are Processed

• Must be folded, altered molecularly biologically active

• REMEMBER: folding, non-covalent interactions among 1o structure aa’s/funct’l grps proper 2o, 3o, 4o structures functional protein

• Post-translational modifications

– Terminal aa’s modified

• fMet cleaved or its formyl grp cleaved from amino terminus

– Terminal aa’s used for signaling cleaved

Post-translational modifications

– Individual aa’s covalently modified

• Phosph’n

Post-translational modifications

– Individual aa’s covalently modified

• Carboxyl grps added

• Methyl grps added

Post-Translational Modifications

– Carbohydrates, isoprenes added

glycoproteins, lipoproteins

– Prosthetic grps added

• Ex: heme added to hemoglobin, cytochromes

– Proteolytic processing

• Polypeptide chains cleaved funct’l proteins

Eukaryotic Translation

• REMEMBER: DNA more complicated, mRNA processed

• One processing step = 5’ cap, polyA tail

– Play a role in complexing of mRNA to ribosome (27-24)

• More (9) initiation factors (Table 27-9)

– Various roles, some impt to processed mRNA

Fig.27-24