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Three Steps of Translation

Initiation
Elongation
Termination

Stages of translation
Initiation
__ribosome recognition
prokaryotes: mRNA Shine-Delgarno sequence which hydrogen bonds
with rRNA
eukaryotes: ribosome attaches to 5 cap

tRNAmet binds to AUG codon


N-formylmethionine used in prokaryotes, tRNAfMet, later removed
Initiation factors

Elongation: use of elongation factors and GTP


Termination
ribosome pauses at stop codon
release factor binds, complex dissociates

Translation Initiation
Initiation requirements:

mRNA molecule
ribosome
Initiator tRNA
Three initiation factors
GTP
Mg2+

Initiation
Small ribosomal subunit binds to _several initiation
factors_
This complex binds to mRNA
In prokaryotes initiation codon _AUG_ calls for fmet (N-formylmethionine)
Shine-Dalgarno sequence AGGAGG precedes start
codon in mRNA; complementary sequence on 3of
16S rRNA __facilitates initiation____
Initiation protein enhances binding of f-met tRNA to
the small subunit sets the reading frame
initiation complex

TRANSLATION INITIATION:
Bacteria
5

Ribosome
binding site
AGGAGGU

Initiation
codon
AUG

3-10 nucleotides

Translation factors:
-initiation factors (IF-1, IF-2, IF-3)
-elongation factors (EF-Tu, EF-Ts,EF-G)
-release factors (RF-1, RF-2, RF-3)
-ribosome recycling factor (RRF)

Initiation Complex
30S small subunit binds __IF1, IF2, IF3
30S +IFs binds to mRNA AUG codon
region
A sequence upstream of AUG is necessary, -810 position
5end of 5UTR ribosome-binding site RBS
(Shine-Delgarno sequence AGGAG purinerich, complementary to UCCUCC pyrimidine
rich region at 3-end of 16S rRNA)

Initiation Complex
Initiator tRNA binds to AUG start codon
Initiator fMet
Methionyl-tRNA synthetase add methionine to
tRNA
Transformylase adds formyl group to methionine
fMet-tRNA.fMet

Initiation Complex
fMet-tRNA binds to AUG of 30S-mRNA complex,
IF3 is released
50S subunit binds
GTP hydrolysis
IF1, IF2 released

Final complex 70S initiation complex


Has three binding sites for aminoacyl-tRNA
E exit
P peptidyl
A aminoacyl

Translation Initiation In Bacteria

PowerPoint Layered Art

Elongation In Prokaryotes
Anticodon of fMet-tRNA is hydrogen-bonded to
AUG in P site
Next codon is in A site
Complementary tRNA binds to A site
tRNA is bound to protein elongator factor EF-Tu
aa-tRNA binds to A site, GTP hydrolysis, release
of EF-Tu-GDP (recycling)

Elongation
Aminoacyl tRNA binds to A site
Growing chain of polypeptide is transferred from
tRNA in P site to tRNA in A site by forming a new
peptide bond
Ribosome translocate along mRNA to position
next codon against A site
tRNA translocate from A-P to P-E sites
Elongation factor EF-TuGTP provide energy

Translocation

Ribosome moves one codon towards _3 end_


Require activity of protein elongation factor EF-G
EF-G-GTP binds to the ribosome, GTP is hydrolyzed
Uncharged tRNA is displaced away from P site,
binds to E site, blocks neat aa-tRNA from binding to
A-site until translocation is complete and peptidyltRNA is bound in P site
After translocation
EF-G is released using GTP hydrolysis and recycled

Termination
Chain-termination codons UAA, UAG, UGA enter A
site
Recognized by release factors RFs (Rf1 and Rf2; only
one in eukaryotes eRF)
Presence of release factor causes the activity change
of peptidyl transferase, add water to carboxyl of
polypeptide chain
Reaction releases polypeptide from tRNA in the P site
Free tRNA moves to E site, mRNA released, ribosome
dissociate

Termination
,
Ribosome Recycling Factor
Elongation Factor G (EF-G)
function to release mRNA and tRNAs from
ribosomes and dissociate the 70s ribosomes
into the 30s and 50s subunits.

Termination
IF-3 also helps the ribosome-recycling
process by converting transiently
dissociated subunits into stable ones by
binding to __30S subunits???_.
This "recycles" the ribosomes for additional
rounds of translation

PowerPoint Layered Art

Ribozymes
A catalytic RNA RNA enzyme
16S rRNA catalysis of peptide bond
Release factor RNA molecule similar to
tRNA
Ribonuclease P - that cleaves the head (5')
end of the precursors of transfer RNA
(tRNA) molecules
snRNPs -

Translation In Eukaryotes
Similar, more __more initiation factors_
tRNA Met is not fMet tRNAs coded by
two different genes
Initiation complex starts at AUG closest to
the 5end
Optimal initiation sequence 5GCC(A/G)CCAUGC-3
G increase efficiency, Kozak rules

Translation In Eukaryotes
tRNAiMet + IF----- P site directly
5cap binding protein CBP (part of eIF-4F initiation
complex) binds to 7-metG at 5end on mRNA
Other IFs bind to CBP-mRNA complex joined by
40S subunit of ribosome
Initiation complex moves 5to 3searching for AUG
( almost always first AUG at 5end)
AUG found IFs released ( except eIF-4F), 60S
binds, complete 80S ribosome

Translation In Eukaryotes
80S initiation complex with Met-tRNA
bound to mRNA with AUG in P site
polyA tail is bound by poly(A)binding
protein (PABP), binds to eIF-4G
Initiation of translation is stimulated

Interaction of 3' and 5' ends of mRNA


important in initiation of translation.

Translation
Prokaryotes
ribosomes are smaller
translation is coupled to
transcription
more than one gene may be
translated from the same
mRNA
translation is polycistronic
the Shine-Delgarno sequence
aligns mRNA on ribosome
the first amino acid
incorporated is Nformylmethionine

Eukaryotes
translation monocistronic
ribosomes recognize
mRNA by binding to 7methylguanosine cap at 5'
end of mRNA
methionine is always the
first amino acid
incorporated

Mutations

Mutations
Change in genetic material -a mutation is an
__alteration in DNA sequence_.
Source of genetic variability
Novel phenotype _mutant_

Mutations
Change in chromosome __number_
Change in chromosome _structure_
Change in the structure of individual gene
__point mutations__

Mutations
Somatic or germinal
Somatic: melanoma - cannot be passed down
generations
Germinal: xray affects germ cells you wouldnt be
affected children will be

Spontaneous or induced
Reversible: happens more often in prokaryotes
prototrophs or auxotrophs
Gain of function, loss of function, neutral

Point Mutations

Addition
Deletion
Substitution
Frame-shift

Missense
Nonsense
Neutral
silent