TranslationTranslation

Function of 5Function of 5′ ′ CAPCAP

Protect mRNA from degradation-RNAses Protect mRNA from degradation-RNAses cannot cleave triphosphate linkagescannot cleave triphosphate linkages

Enhance the translatability of mRNAs-cap Enhance the translatability of mRNAs-cap needed for binding of cap binding protein needed for binding of cap binding protein which is needed for attachment to ribosomewhich is needed for attachment to ribosome

Enhance the transport of the mRNA from Enhance the transport of the mRNA from nucleus to cytoplasmnucleus to cytoplasm

Enhance the splicing of the mRNAsEnhance the splicing of the mRNAs

Function of Poly (A) tailFunction of Poly (A) tail

Protection of mRNAProtection of mRNA

Translatability of mRNA- binding of poly (A)-binding Translatability of mRNA- binding of poly (A)-binding protein I to the poly(A) tail region boosts the protein I to the poly(A) tail region boosts the efficiency with which DNA is translated This protein efficiency with which DNA is translated This protein in turn bind to a translation initiation factor which in turn bind to a translation initiation factor which binds to the cap binding protein attached to the binds to the cap binding protein attached to the cap, effectively linking the 5’ end of the molecule cap, effectively linking the 5’ end of the molecule to its 3’ end. This looped mRNA is more stable and to its 3’ end. This looped mRNA is more stable and readily translated.readily translated.

Efficient transport from the nucleus to cytoplasm Efficient transport from the nucleus to cytoplasm

PolyadenylationPolyadenylation Here, a multi-protein complex cleaves the 3'-most part of a Here, a multi-protein complex cleaves the 3'-most part of a

newly produced RNA and polyadenylates the end produced by newly produced RNA and polyadenylates the end produced by this cleavage. this cleavage.

The cleavage is catalysed by the enzyme The cleavage is catalysed by the enzyme Cleavage and Cleavage and polyadenylation specificity factorpolyadenylation specificity factor (CPSF) and occurs 10–30 (CPSF) and occurs 10–30 nucleotides downstream of its binding site. nucleotides downstream of its binding site.

This site is often the sequence AAUAAA on the RNA, but This site is often the sequence AAUAAA on the RNA, but variants of it exist that bind more weakly to CPSF.variants of it exist that bind more weakly to CPSF.

Two other proteins add specificity to the binding to an RNA: Two other proteins add specificity to the binding to an RNA: CstF and CFI. CstF binds to a GU-rich region further CstF and CFI. CstF binds to a GU-rich region further downstream of CPSF's site.CFI recognises a third site on the downstream of CPSF's site.CFI recognises a third site on the RNA (a set of UGUAA sequences in mammals) and can recruit RNA (a set of UGUAA sequences in mammals) and can recruit CPSF even if the AAUAAA sequence is missing.CPSF even if the AAUAAA sequence is missing.

The polyadenylation signal – the sequence motif recognised by The polyadenylation signal – the sequence motif recognised by the RNA cleavage complex – varies between groups of the RNA cleavage complex – varies between groups of eukaryotes. eukaryotes.

The RNA is cleaved right after transcriptionThe RNA is cleaved right after transcription

When the RNA is cleaved, polyadenylation starts, When the RNA is cleaved, polyadenylation starts, catalysed by polyadenylate polymerase. catalysed by polyadenylate polymerase.

Polyadenylate polymerase builds the poly(A) tail Polyadenylate polymerase builds the poly(A) tail by adding adenosine monophosphate units from by adding adenosine monophosphate units from adenosine triphosphate to the RNA, cleaving off adenosine triphosphate to the RNA, cleaving off pyrophosphate. pyrophosphate.

Another protein, PAB2, binds to the new, short Another protein, PAB2, binds to the new, short poly(A) tail and increases the affinity of poly(A) tail and increases the affinity of polyadenylate polymerase for the RNA. polyadenylate polymerase for the RNA.

When the poly(A) tail is approximately 250 When the poly(A) tail is approximately 250 nucleotides long the enzyme can no longer bind nucleotides long the enzyme can no longer bind to CPSF and polyadenylation stops, thus to CPSF and polyadenylation stops, thus determining the length of the poly(A) tail determining the length of the poly(A) tail

Translation ensures that:Translation ensures that:

polypeptide bonds are formed between polypeptide bonds are formed between adjacent amino acid adjacent amino acid

that the amino acids are linked in the correct that the amino acids are linked in the correct sequence specified by the codons in mRNA.sequence specified by the codons in mRNA.

mRNAs are read in the 5’mRNAs are read in the 5’→3’ direction.→3’ direction.

Proteins are made in the amino to Proteins are made in the amino to carboxyl direction, hence the amino carboxyl direction, hence the amino terminal amino acid is added first.terminal amino acid is added first.

TThe genetic code is a set of three –base he genetic code is a set of three –base code words, called codons, in mRNA that code words, called codons, in mRNA that instruct the ribosome to incorporate specific instruct the ribosome to incorporate specific amino acids into polypeptides. amino acids into polypeptides.

Each base is part of only one codon.Each base is part of only one codon.

There are 64 codons in all.There are 64 codons in all.

Three are stop signals and the rest code for Three are stop signals and the rest code for amino acids.amino acids.

Since there are only 20 amino acids then the code Since there are only 20 amino acids then the code is degenerate. This is made possible by:is degenerate. This is made possible by:

Isoaccepting tRNAs that bind the same amino Isoaccepting tRNAs that bind the same amino acid although they have different anticodons.acid although they have different anticodons.

The third base in a codon is allowed to move The third base in a codon is allowed to move slightly from its normal position to form a non- slightly from its normal position to form a non- Watson-Crick pair with the anticodon. Watson-Crick pair with the anticodon.

This allows the same aminoacyl-tRNA to pair with This allows the same aminoacyl-tRNA to pair with more than one codon. more than one codon.

This is called wobble.This is called wobble.

Is the Genetic code Is the Genetic code Universal?Universal?

The genetic code is not strictly The genetic code is not strictly universal (same codons encoding the universal (same codons encoding the same information in all species).same information in all species).

Termination codons in the standard genetic Termination codons in the standard genetic code can code for aa like tryptophan and code can code for aa like tryptophan and glutamine in some species.glutamine in some species.

Two events occur before protein synthesis:Two events occur before protein synthesis:

Aminacyl-tRNA synthetases join amino acids to Aminacyl-tRNA synthetases join amino acids to their respective tRNAs: this is done in 2 stepstheir respective tRNAs: this is done in 2 steps

First activation of the amino acid with AMP.First activation of the amino acid with AMP.

Secondly tRNAs picks up the activated Secondly tRNAs picks up the activated amino acids. The resulting complexes called amino acids. The resulting complexes called aminoacyl-tRNAs are able to bind to the aminoacyl-tRNAs are able to bind to the mRNA coding sequences so as to align the mRNA coding sequences so as to align the amino acid in the correct order to form the amino acid in the correct order to form the polypeptide chain polypeptide chain

Second ribosomes dissociate into subunits Second ribosomes dissociate into subunits (happens at the end of each translation event).(happens at the end of each translation event).

Diagrammatic representation Diagrammatic representation of tRNA moleculeof tRNA molecule

Properties of tRNA molecule:Properties of tRNA molecule:

An amino acid is attached to transfer RNA An amino acid is attached to transfer RNA before becoming incorporated into a before becoming incorporated into a polypeptide.polypeptide.

tRNAs are responsible for aligning the amino tRNAs are responsible for aligning the amino acids in the correct sequence. acids in the correct sequence.

Each kind of tRNA binds to a specific aa. Each kind of tRNA binds to a specific aa.

It must have an anticodon, a specific It must have an anticodon, a specific complementary binding sequence for the complementary binding sequence for the correct mRNA codon.correct mRNA codon.

It must be recognized by a specific It must be recognized by a specific aminoacyl-tRNA synthase that adds the aminoacyl-tRNA synthase that adds the correct aa.correct aa.

It must have a region for the attachment of the It must have a region for the attachment of the specific aa specified by the anticodon.specific aa specified by the anticodon.

It must be recognized by ribosomes.It must be recognized by ribosomes.

The tRNAs are polynucleotide chains 70 to 80 The tRNAs are polynucleotide chains 70 to 80 nucleotides long, each with several unique nucleotides long, each with several unique basesbases

Complementary base pairing within each tRNA Complementary base pairing within each tRNA molecules causes it to be doubled back and molecules causes it to be doubled back and folded. folded.

Three or more loops of unpaired nucleotides Three or more loops of unpaired nucleotides are formed, one of which contains the are formed, one of which contains the anticodon. anticodon.

The aa binding site is at the 3’ end of the The aa binding site is at the 3’ end of the molecule. molecule.

The carboxyl group of the aa is bound to the The carboxyl group of the aa is bound to the exposed 3’ hydroxyl group of the sugar of the exposed 3’ hydroxyl group of the sugar of the terminal nucleotide.terminal nucleotide.

This leaves the amino group of the aa free to This leaves the amino group of the aa free to participate in peptide bonds. participate in peptide bonds.

The pattern of folding results in constant distance The pattern of folding results in constant distance between the anticodon and the aa, allowing for between the anticodon and the aa, allowing for precise positioning of the aa during translation. precise positioning of the aa during translation.

RibosomesRibosomes Made up of Made up of 65% ribosomal RNA and 35% ribosomal 65% ribosomal RNA and 35% ribosomal

proteins proteins arranged into small and large subunitsarranged into small and large subunits..

Ribosomes consist of two subunits that fit together Ribosomes consist of two subunits that fit together and work as one to translate the mRNA into a and work as one to translate the mRNA into a polypeptide chain during protein synthesis. polypeptide chain during protein synthesis.

The active part of the ribosome is RNAThe active part of the ribosome is RNA

Eukaryotes Ribosomes:Eukaryotes Ribosomes:

Eukaryotes have 80S ribosomes, each consisting of a Eukaryotes have 80S ribosomes, each consisting of a small (40S) and large (60S) subunit. small (40S) and large (60S) subunit.

Their large subunit is composed of a 5S RNA (120 Their large subunit is composed of a 5S RNA (120 nucleotides), a 28S RNA (4700 nucleotides), a 5.8S subunit nucleotides), a 28S RNA (4700 nucleotides), a 5.8S subunit (160 nucleotides) and ~49 proteins. (160 nucleotides) and ~49 proteins.

The small subunit has a 1900 nucleotide (18S) RNA and The small subunit has a 1900 nucleotide (18S) RNA and ~33 proteins ~33 proteins

The S number given each type of rRNA reflects the The S number given each type of rRNA reflects the rate at which the molecules sediment in the rate at which the molecules sediment in the ultracentrifuge. The larger the number, the larger ultracentrifuge. The larger the number, the larger the molecule (but not proportionally).the molecule (but not proportionally).

The 28S, 18S, and 5.8S molecules are The 28S, 18S, and 5.8S molecules are produced by the processing of a single produced by the processing of a single primary transcript from a cluster of identical primary transcript from a cluster of identical copies of a single gene. The 5S molecules are copies of a single gene. The 5S molecules are produced from a different cluster of identical produced from a different cluster of identical genes.genes.

cluster of identical copies cluster of identical copies of a single geneof a single gene

Prokaryotes Ribosomes:Prokaryotes Ribosomes:

Prokaryotes have 70S ribosomes, each Prokaryotes have 70S ribosomes, each consisting of a small (30S) and a large (50S) consisting of a small (30S) and a large (50S) subunit. subunit.

Their large subunit is composed of a 5S RNA Their large subunit is composed of a 5S RNA subunit (consisting of 120 nucleotides), a subunit (consisting of 120 nucleotides), a 23S RNA subunit (2900 nucleotides) and 34 23S RNA subunit (2900 nucleotides) and 34 proteins. proteins.

The 30S subunit has a 1540 nucleotide RNA The 30S subunit has a 1540 nucleotide RNA subunit (16S) bound to 21 proteins subunit (16S) bound to 21 proteins

Translation in ProkaryotesTranslation in Prokaryotes

Translation is divided into three Translation is divided into three stages:stages:

Initiation Initiation Elongation Elongation Termination. Termination.

TranslationTranslation

The initiation codon in prokaryotes is The initiation codon in prokaryotes is usually AUG. The initiating aminoacyl-tRNA usually AUG. The initiating aminoacyl-tRNA is N-formyl-methionyl-tRNAis N-formyl-methionyl-tRNAff

metmet. .

N-formyl-methionine is the first amino acid N-formyl-methionine is the first amino acid incorporated into the polypeptide chain. incorporated into the polypeptide chain.

A 30S initiation complex is formed from a A 30S initiation complex is formed from a free 30S subunit plus a mRNA and fMet-free 30S subunit plus a mRNA and fMet-tRNAtRNAff

met.met.

The 16S rRNA of the 30S initiation complex The 16S rRNA of the 30S initiation complex first base pairs with a sequence called the first base pairs with a sequence called the Shine-Delgarno sequence upstream from Shine-Delgarno sequence upstream from the initiation codon. the initiation codon.

http://themedicalbiochemistrypage.org/images/shine-http://themedicalbiochemistrypage.org/images/shine-delgarno.jpgdelgarno.jpg

This binding is mediated by IF3 with the This binding is mediated by IF3 with the help of IF2 and IF1.help of IF2 and IF1.

• The initiation complex then slides along the The initiation complex then slides along the mRNA until it reaches the initiation codon. mRNA until it reaches the initiation codon.

• A 30S initiation complex is formed from A 30S initiation complex is formed from a free 30S subunit plus a mRNA and a free 30S subunit plus a mRNA and fMet-tRNAfMet-tRNAff

metmet, GTP, IF1, IF2 and IF3., GTP, IF1, IF2 and IF3.

GTP is hydrolysed after the 50S GTP is hydrolysed after the 50S subunit joins the 30S complex to subunit joins the 30S complex to form the 70S initiation complex.form the 70S initiation complex.

Elongation:Elongation:

Is the addition of other aa to the growing Is the addition of other aa to the growing polypeptide chain. polypeptide chain.

Takes place in three steps:Takes place in three steps:

1.1. EF-Tu with the help of GTP, binds an EF-Tu with the help of GTP, binds an aminoacyl-tRNA aminoacyl-tRNA to the A site by specific base-to the A site by specific base-pairing of its pairing of its anticodon and the anticodon and the complementary mRNA codon. complementary mRNA codon.

The amino group of the aa at the A site is aligned with the The amino group of the aa at the A site is aligned with the carboxyl group of the preceding aa at the P site. carboxyl group of the preceding aa at the P site.

2. 2. Peptidyl transferase forms a peptide bond Peptidyl transferase forms a peptide bond between the between the peptide in the P site and the newly peptide in the P site and the newly arrived aminoacyl-arrived aminoacyl- tRNA in the A site.tRNA in the A site.

In this process the aa that is attached to the P site In this process the aa that is attached to the P site is released from its tRNA and becomes attached to is released from its tRNA and becomes attached to the aminoacyl-tRNA at the A sitethe aminoacyl-tRNA at the A site

Peptidyle transferase activity resides on the 50S Peptidyle transferase activity resides on the 50S subunit (on its 23S rRNA) subunit (on its 23S rRNA)

3. EF-G, with GTP translocates the growing 3. EF-G, with GTP translocates the growing peptidyl-tRNA, with its mRNA codon to the P peptidyl-tRNA, with its mRNA codon to the P site. leaving the A site free for the next site. leaving the A site free for the next aminoacyl-tRNA.aminoacyl-tRNA.

Each translocation moves the mRNA one codon’s Each translocation moves the mRNA one codon’s

length through the ribosome so that the mRNA length through the ribosome so that the mRNA codon specifying the next aa in the polypeptide codon specifying the next aa in the polypeptide chain becomes positioned in the unoccupied A site. chain becomes positioned in the unoccupied A site.

The end of the mRNA which is The end of the mRNA which is transcribed first is also the first to be transcribed first is also the first to be translated. translated.

An average sized protein of about An average sized protein of about 360 aa can be assembled by a 360 aa can be assembled by a prokaryote in 18 seconds and by a prokaryote in 18 seconds and by a eukaryotic cell in little over a minute. eukaryotic cell in little over a minute.

Elongation in eukaryotesElongation in eukaryotes

Elongation in eukaryotes is carried out with Elongation in eukaryotes is carried out with two two

elongation factors: eEF-1 and eEF-2:elongation factors: eEF-1 and eEF-2:

The first is eEF-1, whose α subunit act as The first is eEF-1, whose α subunit act as counterparts to EF-Tu.counterparts to EF-Tu.

The second is eEF-2, the counterpart to The second is eEF-2, the counterpart to prokaryotic EF-G prokaryotic EF-G

Animation of translation in Animation of translation in prokaryotesprokaryotes

http://www.phschool.com/science/biohttp://www.phschool.com/science/biology_place/biocoach/translation/elonglogy_place/biocoach/translation/elong1.html1.html

http://www.chromosome.com/http://www.chromosome.com/DNA_animations/protein.movDNA_animations/protein.mov

ElongationElongation

Accuracy of elongationAccuracy of elongation

Mediated in two ways:Mediated in two ways:

The protein-synthesizing machinery gets rid The protein-synthesizing machinery gets rid of ternary complexes bearing the wrong of ternary complexes bearing the wrong aminoacyl-tRNA before GTP hydrolysis.aminoacyl-tRNA before GTP hydrolysis.

It also eliminates incorrect aminoacyl-tRNA It also eliminates incorrect aminoacyl-tRNA in the proofreading step before its amino in the proofreading step before its amino acid gets incorporated into the polypeptide.acid gets incorporated into the polypeptide.

Termination:Termination:

The synthesis of the polypeptide chain is terminated The synthesis of the polypeptide chain is terminated by release factors that recognize the termination or by release factors that recognize the termination or stop codon at the end of the coding sequence. stop codon at the end of the coding sequence.

Prokaryotic translation termination is mediated by Prokaryotic translation termination is mediated by three factors: RF1, RF2 and RF3. RF1 recognizes three factors: RF1, RF2 and RF3. RF1 recognizes UAA and UGA. RF3 is a GTP-binding protein that UAA and UGA. RF3 is a GTP-binding protein that facilitates binding of RF1 and RF2 to the ribosome.facilitates binding of RF1 and RF2 to the ribosome.

The release factors release the newly formed The release factors release the newly formed protein, the mRNA, and the last tRNA used, protein, the mRNA, and the last tRNA used,

The ribosome dissociates into its two subunits, The ribosome dissociates into its two subunits, which are then reused. which are then reused.

Initiation in EukaryotesInitiation in Eukaryotes Eukaryotic 40S ribosomal subunits and initiator tRNA Eukaryotic 40S ribosomal subunits and initiator tRNA

(tRNA(tRNAiimetmet) locate the appropriate start codon by binding ) locate the appropriate start codon by binding

to the 5to the 5′-cap of an mRNA and scan downstream until ′-cap of an mRNA and scan downstream until they locate the first AUG.they locate the first AUG.

The initiation factors are also different than those in The initiation factors are also different than those in prokaryotic initiation:prokaryotic initiation:

eIF2 is involved in binding Met-eIF2 is involved in binding Met-tRNAtRNAiimet met to the to the

ribosome.ribosome.

eIF2 is a GTP-binding protein responsible for bringing the eIF2 is a GTP-binding protein responsible for bringing the initiator tRNA to the P-site of the pre-initiation complex. initiator tRNA to the P-site of the pre-initiation complex.

It has specificity for the methionine-charged initiator tRNA, It has specificity for the methionine-charged initiator tRNA, which is distinct from other methionine-charged tRNAs which is distinct from other methionine-charged tRNAs specific for elongation of the polypeptide chain. specific for elongation of the polypeptide chain.

Once it has placed the initiator tRNA on the AUG start codon Once it has placed the initiator tRNA on the AUG start codon in the P-site, it hydrolyzes GTP into GDP, and dissociates. in the P-site, it hydrolyzes GTP into GDP, and dissociates.

eIF2B activates eIF2 by replacing its GDP eIF2B activates eIF2 by replacing its GDP with GTP.with GTP.

eIF3 binds to the 40S ribosomal subunit eIF3 binds to the 40S ribosomal subunit and inhibits its reassociation with 60S and inhibits its reassociation with 60S subunit:subunit:

eIF1, eIF1A, and eIF3, all bind to the ribosome eIF1, eIF1A, and eIF3, all bind to the ribosome subunit-mRNA complex. subunit-mRNA complex.

They have been implicated in preventing the They have been implicated in preventing the large ribosomal subunit from binding the small large ribosomal subunit from binding the small subunit before it is ready to commence subunit before it is ready to commence elongationelongation. .

eIF4 is a cap-binding protein composed of eIF4 is a cap-binding protein composed of 3 parts:3 parts:

eIF4E has cap-binding activityeIF4E has cap-binding activity

eIFA has RNA helicase activity and eIFA has RNA helicase activity and unwinds 5’-leaders of eukaryotic unwinds 5’-leaders of eukaryotic mRNAs. mRNAs.

eIF4G is an adaptor protein that bind to eIF4G is an adaptor protein that bind to proteins like: eIF3 (the 40S ribosomal proteins like: eIF3 (the 40S ribosomal subunit binding protein) and Pab1p (a subunit binding protein) and Pab1p (a poly (A) tail binding protein) thereby poly (A) tail binding protein) thereby associating 40S subunit with both ends associating 40S subunit with both ends of mRNA and stimulate initiation.of mRNA and stimulate initiation.

eIF5 encourages association between the eIF5 encourages association between the 43S complex (comprising the 40S subunit 43S complex (comprising the 40S subunit plus Met-plus Met-tRNAtRNAii

metmet) and large ribosomal ) and large ribosomal subunit:subunit:

eIF5A is a GTPase-activating protein, which eIF5A is a GTPase-activating protein, which helps the large ribosomal subunit associate helps the large ribosomal subunit associate with the small subunit. It is required for GTP-with the small subunit. It is required for GTP-hydrolysis by eIF2 and contains the unusual hydrolysis by eIF2 and contains the unusual amino acid hypusine.amino acid hypusine.

eIF5B is a GTPase, and is involved in assembly eIF5B is a GTPase, and is involved in assembly of the full ribosome (which requires GTP of the full ribosome (which requires GTP hydrolysis).hydrolysis).

eIF6 binds to the 60S subunit and blocks eIF6 binds to the 60S subunit and blocks its reassociation with 40S subunit.its reassociation with 40S subunit.

http://wormbook.sanger.ac.uk/chapters/http://wormbook.sanger.ac.uk/chapters/www_mechregultranslation/RhoadsMRTfig1.jpgwww_mechregultranslation/RhoadsMRTfig1.jpg

Termination in EukaryotesTermination in Eukaryotes

Eukaryotes have 2 release factors:Eukaryotes have 2 release factors:

eRF1 that recognizes all three eRF1 that recognizes all three termination codonstermination codons

eRF3, a ribosome-dependent GTPase eRF3, a ribosome-dependent GTPase that helps eRF1 release the finished that helps eRF1 release the finished polypeptide.polypeptide.

PolysomesPolysomes A single mRNA molecule usually has A single mRNA molecule usually has

many ribosomes traveling along it, in many ribosomes traveling along it, in various stages of synthesizing the various stages of synthesizing the polypeptide. This complex is called a polypeptide. This complex is called a polysomepolysome

ReferencesReferences

http://www.frontiers-in-genetics.org/en/pictures/translation_1.jpghttp://www.frontiers-in-genetics.org/en/pictures/translation_1.jpg http://en.wikipedia.org/wiki/Eukaryotic_initiation_factorhttp://en.wikipedia.org/wiki/Eukaryotic_initiation_factor Weaver R. F. Molecular Biology. Fourth edition. McGraw Hill Higher Weaver R. F. Molecular Biology. Fourth edition. McGraw Hill Higher

Education.Education. http://www.uic.edu/classes/bios/bios100/summer2002/ribosome01.gifhttp://www.uic.edu/classes/bios/bios100/summer2002/ribosome01.gif

http://www.agen.ufl.edu/~chyn/age2062/OnLineBiology/OLBB/www.emc.maricopa.edhttp://www.agen.ufl.edu/~chyn/age2062/OnLineBiology/OLBB/www.emc.maricopa.edu/faculty/farabee/BIOBK/ribosome.gifu/faculty/farabee/BIOBK/ribosome.gif

http://en.wikipedia.org/wiki/Ribosomehttp://en.wikipedia.org/wiki/Ribosome