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Protein SynthesisProtein Synthesis(From Nucleus to (From Nucleus to

Cytoplasm)Cytoplasm)

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The Central Dogma The Central Dogma www.video.sina.com.cnwww.video.sina.com.cn

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REPLICATION

TRANSCRIPTIONmRNA

TRANSLATIONPROTEINDNADNA

tRNA Amino Acids

Ribosomes

rRNA Protein

The Central Dogma (in equation form)

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Initial Problem:Initial Problem:DNA codes for proteins BUT DNA is DNA codes for proteins BUT DNA is

confined to the nucleus and the ‘equipment’ to confined to the nucleus and the ‘equipment’ to carry out protein synthesis is in the cytoplasm.carry out protein synthesis is in the cytoplasm.(The equipment includes ribosomes, tRNA, (The equipment includes ribosomes, tRNA, and amino acids).and amino acids).

Solution???Solution???DNA can be “copied” into an RNA molecule DNA can be “copied” into an RNA molecule

((messenger RNAmessenger RNA ( (mRNAmRNA)), which can travel )), which can travel from the nucleus to the cytoplasm carrying the from the nucleus to the cytoplasm carrying the instructions for building a protein.instructions for building a protein.

This DNA to mRNA copying process is known This DNA to mRNA copying process is known as as transcriptiontranscription (see fig. 25.7 on p. 512). (see fig. 25.7 on p. 512).

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Visual Examples of Visual Examples of TranscriptionTranscription

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TranscriptionTranscription During this process, DNA serves as a template During this process, DNA serves as a template

(guide) for the production of mRNA(guide) for the production of mRNA The enzyme The enzyme DNA helicase DNA helicase serves to unwind and serves to unwind and

‘unzip’ the portion of the DNA double helix (ie. The ‘unzip’ the portion of the DNA double helix (ie. The GENE) that is to be transcribed (copied into mRNA).GENE) that is to be transcribed (copied into mRNA).

Once this occurs, free-floating RNA nucleotides Once this occurs, free-floating RNA nucleotides (within the nucleus along with free-floating DNA (within the nucleus along with free-floating DNA nucleotides) bond to the exposed bases of DNA nucleotides) bond to the exposed bases of DNA through through complementary base-pairingcomplementary base-pairing (forming (forming hydrogen bonds).hydrogen bonds).

** RNA nucleotides bind to only ONE exposed DNA strand.** RNA nucleotides bind to only ONE exposed DNA strand.

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AA (mRNA) binds with (mRNA) binds with TT (DNA). (DNA). UU (mRNA) binds with (mRNA) binds with AA (DNA). (DNA). CC (mRNA) binds with (mRNA) binds with GG (DNA). (DNA). G G (mRNA) binds with (mRNA) binds with CC (DNA). (DNA).

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The first three mRNA bases are always The first three mRNA bases are always AUG AUG (called (called the the startstart codon), which means that the first three codon), which means that the first three DNA bases that are transcribed are DNA bases that are transcribed are TACTAC..

The last three mRNA bases are always one of The last three mRNA bases are always one of UAG, UAG, UAA, UAA, or or UGA UGA ((STOPSTOP codons), which means that the codons), which means that the last three DNA bases transcribed are always one of last three DNA bases transcribed are always one of ATC, ATT, ATC, ATT, or or ACT.ACT.

The enzyme The enzyme RNA Polymerase RNA Polymerase then works to join then works to join added mRNA nucleotides to each other (sugar-P-added mRNA nucleotides to each other (sugar-P-sugar-P etc…) through dehydration synthesis sugar-P etc…) through dehydration synthesis (producing water).(producing water).

So, the mRNA nucleotides form spontaneous H-So, the mRNA nucleotides form spontaneous H-bonds with exposed DNA bases but then need bonds with exposed DNA bases but then need enzymatic aid (RNA polymerase) to form the actual enzymatic aid (RNA polymerase) to form the actual mRNA chain.mRNA chain.

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DNA helicase continues to unwind/unzip the DNA helicase continues to unwind/unzip the DNA until the gene that requires copying has DNA until the gene that requires copying has been fully exposed.been fully exposed. The signal for DNA helicase to stop is when it The signal for DNA helicase to stop is when it

encounters encounters ATC, ATTATC, ATT, or , or ACT.ACT.

A A poly-adenine tailpoly-adenine tail is added to one end of is added to one end of the finished mRNA while a guanine-based the finished mRNA while a guanine-based ‘‘capcap’ is added to the other side to protect ’ is added to the other side to protect the molecule from cytoplasmic enzymes.the molecule from cytoplasmic enzymes.

Once this is finished, the mRNA moves into Once this is finished, the mRNA moves into the cytoplasm, through a nuclear pore, and the cytoplasm, through a nuclear pore, and the DNA joins back together by reforming the DNA joins back together by reforming its complementary base-pairing H-bonds.its complementary base-pairing H-bonds.

1010START Codon STOP Codon

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The Genetic CodeThe Genetic Code Query: How is a sequence of nitrogenous bases Query: How is a sequence of nitrogenous bases

on mRNA going to be used to code for a sequence on mRNA going to be used to code for a sequence of amino acids and hence, a protein???of amino acids and hence, a protein???

First of all, DNA is the universal code (ie. Every First of all, DNA is the universal code (ie. Every living thing has DNA, and DNA codes for proteins living thing has DNA, and DNA codes for proteins (through the use of mRNA)).(through the use of mRNA)).

There are 20 amino acids in nature.There are 20 amino acids in nature. There are 4 different nitrogenous bases in both There are 4 different nitrogenous bases in both

DNA and mRNA, and they serve as a ‘code’ for DNA and mRNA, and they serve as a ‘code’ for the amino acid sequence of proteins.the amino acid sequence of proteins.

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The code reads as a TRIPLET CODE (4The code reads as a TRIPLET CODE (433 = 64 = 64 possibilities), meaning that three nitrogenous possibilities), meaning that three nitrogenous bases, as a group, code for ONE amino acid.bases, as a group, code for ONE amino acid.

A singlet code could only code for 4A singlet code could only code for 411 = 4 = 4 amino acids (yet, there are 20) – thus proving amino acids (yet, there are 20) – thus proving inadequate.inadequate.

A doublet code could only code for 4A doublet code could only code for 422 = 16 = 16 amino acids.amino acids.

A group of three mRNA nucleotides (bases) is A group of three mRNA nucleotides (bases) is called a called a CODONCODON. In total, there are 64 (4. In total, there are 64 (433) ) different codons.different codons.

A group of three DNA nucleotides (bases) is A group of three DNA nucleotides (bases) is called a called a DNA triplet. DNA triplet.

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There are 61 different codons that correspond to There are 61 different codons that correspond to the 20 different amino acids (one of these 61 is the 20 different amino acids (one of these 61 is AUGAUG, which is the ‘start’ codon, that codes for the , which is the ‘start’ codon, that codes for the amino acid amino acid methioninemethionine).).

The other three codons are called STOP codons, The other three codons are called STOP codons, which terminate the formation of the which terminate the formation of the polypeptide/protein chain. polypeptide/protein chain.

These STOP codons DO NOT code for an amino These STOP codons DO NOT code for an amino acid. acid.

The Genetic code is sometimes referred to as The Genetic code is sometimes referred to as being being redundantredundant, because most amino acids are , because most amino acids are coded for by 2-6 different codons.coded for by 2-6 different codons.

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AAA GCU ACC GGU UAC GUC UAG AAA GCU ACC GGU UAC GUC UAG mRNA sequencemRNA sequence Questions: i. How many codons present? Questions: i. How many codons present?

ii. How many a. acids coded for? ii. How many a. acids coded for? iii. What is the a. acid sequence?iii. What is the a. acid sequence?

lysine, alanine, threonine, glycine, lysine, alanine, threonine, glycine, tyrosine, valine, STOPtyrosine, valine, STOP

/ / / / / /7

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iv. What was the DNA sequence of iv. What was the DNA sequence of triplets that coded for this mRNA?triplets that coded for this mRNA?TTT CGA TGG CCA ATG CAG ATCTTT CGA TGG CCA ATG CAG ATC

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TranslationTranslation The process of turning mRNA into a protein (ie. The process of turning mRNA into a protein (ie.

Translating the language of nitrogenous bases into Translating the language of nitrogenous bases into the language of amino acids).the language of amino acids).

Recall that mRNA is constructed in the nucleus Recall that mRNA is constructed in the nucleus through the process of transcription, and is sent out through the process of transcription, and is sent out of the nucleus through a nuclear pore.of the nucleus through a nuclear pore.

Once mRNA enters the cytoplasm, it immediately Once mRNA enters the cytoplasm, it immediately associates with a associates with a ribosome ribosome (either a ‘free’ ribosome (either a ‘free’ ribosome or a Rough ER ribosome).or a Rough ER ribosome).

The ribosome attaches to the mRNA at the guanine-The ribosome attaches to the mRNA at the guanine-based ‘cap’ that not only served as protection from based ‘cap’ that not only served as protection from enzymes, but acted as a ‘start here’ signal.enzymes, but acted as a ‘start here’ signal.

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Translation requires two other types of RNA:Translation requires two other types of RNA:i.i. rRNA (Ribosomal RNA)rRNA (Ribosomal RNA)

-- joins with ribosomal proteins (from -- joins with ribosomal proteins (from nucleolus) to form ribosomes.nucleolus) to form ribosomes.-- produced in the -- produced in the nucleolusnucleolus..-- one ribosome has two subunits:-- one ribosome has two subunits:

a. Large subunit (3 rRNAs and proteins)a. Large subunit (3 rRNAs and proteins) b. Small subunit (1 rRNA and proteins)b. Small subunit (1 rRNA and proteins)-- the two subunits remain close together but -- the two subunits remain close together but do not actually attach until just prior to do not actually attach until just prior to translation.translation.-- rRNA is not involved in any coding or -- rRNA is not involved in any coding or translating, it is purely translating, it is purely structural.structural.

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ii. tRNA (Transfer RNA)ii. tRNA (Transfer RNA)-- located in the cytoplasm and serve as -- located in the cytoplasm and serve as

‘carriers’ of singular amino acids to the ‘carriers’ of singular amino acids to the mRNA/ribosome complex.mRNA/ribosome complex.

-- tRNAs carry one amino acid at one end, and a -- tRNAs carry one amino acid at one end, and a specific ANTICODON at the other end.specific ANTICODON at the other end.

-- this -- this anticodonanticodon will ‘match-up’ with a will ‘match-up’ with a complementary codon on mRNA (through complementary codon on mRNA (through complementary base-pairing) (fig. 25.8 p. 513).complementary base-pairing) (fig. 25.8 p. 513).

-- tRNA molecules are very specific (ie. A tRNA -- tRNA molecules are very specific (ie. A tRNA with a certain anticodon will ALWAYS be carrying the with a certain anticodon will ALWAYS be carrying the same amino acid).same amino acid).

-- remember, though, that the translation table is -- remember, though, that the translation table is translated from mRNA codons, not the tRNA translated from mRNA codons, not the tRNA anticodons.anticodons.

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Eg. If the mRNA codon being translated is Eg. If the mRNA codon being translated is ACGACG, , what anticodon and amino acid will the tRNA what anticodon and amino acid will the tRNA molecule, specific to this codon, be carrying?molecule, specific to this codon, be carrying?

Ans. Anticodon = UGCAns. Anticodon = UGC Amino Acid = Threonine (need table)Amino Acid = Threonine (need table)

Eg 2. Codon = Eg 2. Codon = CAACAA, find anticodon and a. acid., find anticodon and a. acid.Anticodon = GUU, A. acid = GlutamineAnticodon = GUU, A. acid = Glutamine

Eg 3. DNA triplet = Eg 3. DNA triplet = TTCTTC, find mRNA codon, , find mRNA codon, tRNA anticodon, and amino acid.tRNA anticodon, and amino acid.

mRNA codon = mRNA codon = AAG, AAG, anticodon = UUCanticodon = UUCAmino acid = LysineAmino acid = Lysine

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HINT: The anticodon will be the same as HINT: The anticodon will be the same as the original DNA triplet except that a U the original DNA triplet except that a U will replace a T.will replace a T.

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Three Steps of Three Steps of TranslationTranslation

1.1. INITIATIONINITIATION-- the ‘cap’ of mRNA binds to the ribosome and -- the ‘cap’ of mRNA binds to the ribosome and

the ribosome moves along the mRNA, the ribosome moves along the mRNA, ‘reading’ it, until it comes upon the ‘start’ ‘reading’ it, until it comes upon the ‘start’ codon, codon, AUGAUG..

-- the tRNA with anticodon UAC binds to the -- the tRNA with anticodon UAC binds to the AUGAUG codon (through complementary base-pairing) codon (through complementary base-pairing) at the A-site, and delivers the first amino acid at the A-site, and delivers the first amino acid Methionine.Methionine.

-- see handout (crude handout, that is)-- see handout (crude handout, that is)

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A-site

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2.2. ELONGATIONELONGATION (lengthening of the amino acid (lengthening of the amino acid chain).chain).

-- firstly, the ribosome is large enough to -- firstly, the ribosome is large enough to accommodate two tRNAs at the same time accommodate two tRNAs at the same time the ‘incoming’ tRNA (at the A-site) and the the ‘incoming’ tRNA (at the A-site) and the ‘outgoing’ tRNA (at the P-site).‘outgoing’ tRNA (at the P-site).

-- after the first tRNA binds to the mRNA codon, -- after the first tRNA binds to the mRNA codon, the ribosome shifts one codon (3 bases), thus the ribosome shifts one codon (3 bases), thus exposing a new codon in the A-site which can exposing a new codon in the A-site which can then be bonded to by a new tRNA with the then be bonded to by a new tRNA with the complementary anticodon.complementary anticodon.

-- after this next tRNA binds, the ribosome shifts -- after this next tRNA binds, the ribosome shifts again and ‘bumps’ the tRNA in the P-site off of again and ‘bumps’ the tRNA in the P-site off of the ribosome.the ribosome.

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-- before it is bumped off of the ribosome’s P-site, -- before it is bumped off of the ribosome’s P-site, the outgoing tRNA molecule always passes the the outgoing tRNA molecule always passes the amino acid chain, via dehydration synthesis, to amino acid chain, via dehydration synthesis, to the tRNA that is shifting from the A-site to the the tRNA that is shifting from the A-site to the P-site.P-site.

-- the liberated or bumped tRNA (now without an -- the liberated or bumped tRNA (now without an amino acid) will eventually pick up the same amino acid) will eventually pick up the same amino acid that it just ceded and will rejoin the amino acid that it just ceded and will rejoin the group of tRNAs waiting to be ‘chosen’.group of tRNAs waiting to be ‘chosen’.

-- amino acids are readily available in the -- amino acids are readily available in the cytoplasm.cytoplasm.

-- the ribosome continually shifts to accept more -- the ribosome continually shifts to accept more tRNA molecules so that the protein chain can tRNA molecules so that the protein chain can grow one amino acid at a time.grow one amino acid at a time.

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P-site A-site

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iii. iii. TERMINATIONTERMINATION-- -- occurs once the codon appearing in the A-site of the occurs once the codon appearing in the A-site of the

ribosome is a STOP codon.ribosome is a STOP codon.-- this STOP codon is recognized by the ribosome complex -- this STOP codon is recognized by the ribosome complex

and a RELEASE FACTOR protein is summoned from and a RELEASE FACTOR protein is summoned from the cytoplasm into the A-site.the cytoplasm into the A-site.

-- there is NO tRNA molecule for these codons.-- there is NO tRNA molecule for these codons.-- once the release factor protein binds to the STOP codon, -- once the release factor protein binds to the STOP codon,

the ribosome dissociates into its two subunits and falls the ribosome dissociates into its two subunits and falls off the mRNA (which is recycled).off the mRNA (which is recycled).

-- the peptide/protein chain is released by the tRNA in the -- the peptide/protein chain is released by the tRNA in the P-site into the lumen of the Rough ER (if for export), or P-site into the lumen of the Rough ER (if for export), or into a transition vesicle bound for the Golgi for into a transition vesicle bound for the Golgi for modifications (if it is to remain in the cell).modifications (if it is to remain in the cell).

-- see fig. 25.9 p. 514, fig 25.11 p. 516-- see fig. 25.9 p. 514, fig 25.11 p. 516-- read translation summary on pp. 512-516.-- read translation summary on pp. 512-516.

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By simply eating protein, you can build new, custom proteins and maybe look like this:

Yes, and once again, I am Hans; and I am Franz; and we want to PUMP…YOU UP! Especially you ‘girly’ men!