Transcript Biochemistry Lecture 23 THE LAST ONE!

Translation
Chapter 27
Central Dogma
• Genetic code contained w/in 4 deoxynucleotide 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: Nformylmethionine
(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
 a-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