Transcript Translation - UtechDMD2015

Translation
Function of 5′ CAP
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Protect mRNA from degradation-RNAses cannot
cleave triphosphate linkages
Enhance the translatability of mRNAs-cap needed
for binding of cap binding protein which is needed
for attachment to ribosome
Enhance the transport of the mRNA from nucleus
to cytoplasm
Enhance the splicing of the mRNAs
Function of Poly (A) tail
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Protection of mRNA
Translatability of mRNA- binding of poly (A)-binding
protein I to the poly(A) tail region boosts the efficiency
with which DNA is translated This protein in turn bind to
a translation initiation factor which binds to the cap
binding protein attached to the cap, effectively linking
the 5’ end of the molecule to its 3’ end. This looped
mRNA is more stable and readily translated.
Efficient transport from the nucleus to cytoplasm
Polyadenylation
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Here, a multi-protein complex cleaves the 3'-most part of a
newly produced RNA and polyadenylates the end produced by
this cleavage.
The cleavage is catalysed by the enzyme Cleavage and
polyadenylation specificity factor (CPSF) and occurs 10–30
nucleotides downstream of its binding site.
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This site is often the sequence AAUAAA on the RNA, but
variants of it exist that bind more weakly to CPSF.
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Two other proteins add specificity to the binding to an RNA:
CstF and CFI. CstF binds to a GU-rich region further
downstream of CPSF's site.CFI recognises a third site on the
RNA (a set of UGUAA sequences in mammals) and can recruit
CPSF even if the AAUAAA sequence is missing.
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The polyadenylation signal – the sequence motif recognised by
the RNA cleavage complex – varies between groups of
eukaryotes.
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The RNA is cleaved right after transcription
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When the RNA is cleaved, polyadenylation starts,
catalysed by polyadenylate polymerase.
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Polyadenylate polymerase builds the poly(A) tail by
adding adenosine monophosphate units from adenosine
triphosphate to the RNA, cleaving off pyrophosphate.
Another protein, PAB2, binds to the new, short poly(A)
tail and increases the affinity of polyadenylate
polymerase for the RNA.
When the poly(A) tail is approximately 250 nucleotides
long the enzyme can no longer bind to CPSF and
polyadenylation stops, thus determining the length of
the poly(A) tail
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Translation ensures that:
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polypeptide bonds are formed between adjacent
amino acid
that the amino acids are linked in the correct
sequence specified by the codons in mRNA.
mRNAs are read in the 5’→3’ direction.
Proteins are made in the amino to carboxyl direction,
hence the amino terminal amino acid is added first.
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The genetic code is a set of three –base code
words, called codons, in mRNA that instruct the
ribosome to incorporate specific amino acids into
polypeptides.
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Each base is part of only one codon.
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There are 64 codons in all.
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Three are stop signals and the rest code for amino
acids.
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Since there are only 20 amino acids then the code is
degenerate. This is made possible by:
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Isoaccepting tRNAs that bind the same amino acid
although they have different anticodons.
The third base in a codon is allowed to move slightly
from its normal position to form a non- Watson-Crick
pair with the anticodon.
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This allows the same aminoacyl-tRNA to pair with more than
one codon.
This is called wobble.
Is the Genetic code Universal?
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The genetic code is not strictly universal
(same codons encoding the same
information in all species).
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Termination codons in the standard genetic
code can code for aa like tryptophan and
glutamine in some species.
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Two events occur before protein synthesis:
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Aminacyl-tRNA synthetases join amino acids to their
respective tRNAs: this is done in 2 steps
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First activation of the amino acid with AMP.
Secondly tRNAs picks up the activated amino
acids. The resulting complexes called aminoacyltRNAs are able to bind to the mRNA coding
sequences so as to align the amino acid in the
correct order to form the polypeptide chain
Second ribosomes dissociate into subunits (happens
at the end of each translation event).
Diagrammatic representation of
tRNA molecule
Properties of tRNA molecule:
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An amino acid is attached to transfer RNA before
becoming incorporated into a polypeptide.
tRNAs are responsible for aligning the amino
acids in the correct sequence.
Each kind of tRNA binds to a specific aa.
It must have an anticodon, a specific
complementary binding sequence for the correct
mRNA codon.
It must be recognized by a specific aminoacyltRNA synthase that adds the correct aa.
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It must have a region for the attachment of the
specific aa specified by the anticodon.
It must be recognized by ribosomes.
The tRNAs are polynucleotide chains 70 to 80
nucleotides long, each with several unique bases
Complementary base pairing within each tRNA
molecules causes it to be doubled back and
folded.
Three or more loops of unpaired nucleotides are
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The aa binding site is at the 3’ end of the molecule.
The carboxyl group of the aa is bound to the
exposed 3’ hydroxyl group of the sugar of the
terminal nucleotide.
This leaves the amino group of the aa free to
participate in peptide bonds.
The pattern of folding results in constant distance
between the anticodon and the aa, allowing for
precise positioning of the aa during translation.
Ribosomes
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Made up of 65% ribosomal RNA and 35% ribosomal proteins
arranged into small and large subunits.
Ribosomes consist of two subunits that fit together and work
as one to translate the mRNA into a polypeptide chain during
protein synthesis.
The active part of the ribosome is RNA
Eukaryotes Ribosomes:
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Eukaryotes have 80S ribosomes, each consisting of a small (40S)
and large (60S) subunit.
Their large subunit is composed of a 5S RNA (120 nucleotides), a
28S RNA (4700 nucleotides), a 5.8S subunit (160 nucleotides) and
~49 proteins.
The small subunit has a 1900 nucleotide (18S) RNA and ~33
proteins
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The S number given each type of rRNA reflects the rate at
which the molecules sediment in the ultracentrifuge. The
larger the number, the larger the molecule (but not
proportionally).
The 28S, 18S, and 5.8S molecules are produced by
the processing of a single primary transcript from a
cluster of identical copies of a single gene. The 5S
molecules are produced from a different cluster of
identical genes.
cluster of identical copies of a
single gene
Prokaryotes Ribosomes:
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Prokaryotes have 70S ribosomes, each
consisting of a small (30S) and a large (50S)
subunit.
Their large subunit is composed of a 5S RNA
subunit (consisting of 120 nucleotides), a 23S
RNA subunit (2900 nucleotides) and 34 proteins.
The 30S subunit has a 1540 nucleotide RNA
subunit (16S) bound to 21 proteins
Translation in Prokaryotes
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Translation is divided into three stages:
Initiation
Elongation
Termination.
Translation
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The initiation codon in prokaryotes is usually AUG.
The initiating aminoacyl-tRNA is N-formylmethionyl-tRNAfmet.
N-formyl-methionine is the first amino acid
incorporated into the polypeptide chain.
A 30S initiation complex is formed from a free 30S
subunit plus a mRNA and fMet-tRNAfmet.
The 16S rRNA of the 30S initiation complex first
base pairs with a sequence called the ShineDelgarno sequence upstream from the initiation
codon.
http://themedicalbiochemistrypage.org/images/shine-delgarno.jpg
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This binding is mediated by IF3 with the
help of IF2 and IF1.
• The initiation complex then slides along the
mRNA until it reaches the initiation codon.
• A 30S initiation complex is formed from a
free 30S subunit plus a mRNA and fMettRNAfmet, GTP, IF1, IF2 and IF3.
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GTP is hydrolysed after the 50S subunit
joins the 30S complex to form the 70S
initiation complex.
Elongation:
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Is the addition of other aa to the growing
polypeptide chain.
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Takes place in three steps:
1. EF-Tu with the help of GTP, binds an aminoacyl-tRNA to
the A site by specific base-pairing of its anticodon and
the complementary mRNA codon.
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2.
The amino group of the aa at the A site is aligned with the
carboxyl group of the preceding aa at the P site.
Peptidyl transferase forms a peptide bond between the
peptide in the P site and the newly arrived aminoacyltRNA in the A site.
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In this process the aa that is attached to the P site is
released from its tRNA and becomes attached to the
aminoacyl-tRNA at the A site
Peptidyle transferase activity resides on the 50S subunit
(on its 23S rRNA)
3. EF-G, with GTP translocates the growing peptidyltRNA, with its mRNA codon to the P site. leaving
the A site free for the next aminoacyl-tRNA.
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Each translocation moves the mRNA one codon’s length
through the ribosome so that the mRNA codon
specifying the next aa in the polypeptide chain
becomes positioned in the unoccupied A site.
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The end of the mRNA which is transcribed
first is also the first to be translated.
An average sized protein of about 360 aa
can be assembled by a prokaryote in 18
seconds and by a eukaryotic cell in little
over a minute.
Elongation in eukaryotes
Elongation in eukaryotes is carried out with two
elongation factors: eEF-1 and eEF-2:
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The first is eEF-1, whose α subunit act as
counterparts to EF-Tu.
The second is eEF-2, the counterpart to
prokaryotic EF-G
Animation of translation in
prokaryotes
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http://www.phschool.com/science/biology
_place/biocoach/translation/elong1.html
http://www.chromosome.com/DNA_anima
tions/protein.mov
Elongation
Accuracy of elongation
Mediated in two ways:
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The protein-synthesizing machinery gets rid of
ternary complexes bearing the wrong aminoacyltRNA before GTP hydrolysis.
It also eliminates incorrect aminoacyl-tRNA in
the proofreading step before its amino acid gets
incorporated into the polypeptide.
Termination:
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The synthesis of the polypeptide chain is terminated by
release factors that recognize the termination or stop codon
at the end of the coding sequence.
Prokaryotic translation termination is mediated by three
factors: RF1, RF2 and RF3. RF1 recognizes UAA and UGA.
RF3 is a GTP-binding protein that facilitates binding of RF1
and RF2 to the ribosome.
The release factors release the newly formed protein, the
mRNA, and the last tRNA used,
The ribosome dissociates into its two subunits, which are
then reused.
Initiation in Eukaryotes
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Eukaryotic 40S ribosomal subunits and initiator tRNA (tRNAimet)
locate the appropriate start codon by binding to the 5′-cap of
an mRNA and scan downstream until they locate the first AUG.
The initiation factors are also different than those in prokaryotic
initiation:
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eIF2 is involved in binding Met-tRNAimet to the ribosome.
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eIF2 is a GTP-binding protein responsible for bringing the initiator
tRNA to the P-site of the pre-initiation complex.
It has specificity for the methionine-charged initiator tRNA, which is
distinct from other methionine-charged tRNAs specific for elongation
of the polypeptide chain.
Once it has placed the initiator tRNA on the AUG start codon in the
P-site, it hydrolyzes GTP into GDP, and dissociates.
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eIF2B activates eIF2 by replacing its GDP with
GTP.
eIF3 binds to the 40S ribosomal subunit and
inhibits its reassociation with 60S subunit:
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eIF1, eIF1A, and eIF3, all bind to the ribosome
subunit-mRNA complex.
They have been implicated in preventing the large
ribosomal subunit from binding the small subunit
before it is ready to commence elongation.
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eIF4 is a cap-binding protein composed of 3
parts:
 eIF4E
has cap-binding activity
 eIFA
has RNA helicase activity and unwinds
5’-leaders of eukaryotic mRNAs.
 eIF4G
is an adaptor protein that bind to
proteins like: eIF3 (the 40S ribosomal
subunit binding protein) and Pab1p (a poly
(A) tail binding protein) thereby associating
40S subunit with both ends of mRNA and
stimulate initiation.
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eIF5 encourages association between the 43S
complex (comprising the 40S subunit plus MettRNAimet) and large ribosomal subunit:
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eIF5A is a GTPase-activating protein, which helps
the large ribosomal subunit associate with the small
subunit. It is required for GTP-hydrolysis by eIF2
and contains the unusual amino acid hypusine.
eIF5B is a GTPase, and is involved in assembly of
the full ribosome (which requires GTP hydrolysis).
eIF6 binds to the 60S subunit and blocks its
reassociation with 40S subunit.
http://wormbook.sanger.ac.uk/chapters/www_mechregultranslation/Rh
oadsMRTfig1.jpg
Termination in Eukaryotes
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Eukaryotes have 2 release factors:
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eRF1 that recognizes all three termination
codons
eRF3, a ribosome-dependent GTPase that
helps eRF1 release the finished polypeptide.
Polysomes
 A single mRNA molecule usually has many
ribosomes traveling along it, in various
stages of synthesizing the polypeptide.
This complex is called a polysome
References
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http://www.frontiers-in-genetics.org/en/pictures/translation_1.jpg
http://en.wikipedia.org/wiki/Eukaryotic_initiation_factor
Weaver R. F. Molecular Biology. Fourth edition. McGraw Hill Higher Education.
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http://www.uic.edu/classes/bios/bios100/summer2002/ribosome01.gif
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http://www.agen.ufl.edu/~chyn/age2062/OnLineBiology/OLBB/www.emc.maricopa.edu/faculty/fa
rabee/BIOBK/ribosome.gif
http://en.wikipedia.org/wiki/Ribosome