Chapter 7 - DNA to Protein_2

8/13/2019 Chapter 7 - DNA to Protein_2

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From DNA to Protein

BYROSDIANA NATZIR

Information

INFORMATION GENETICS

DEPARTMENT OF BIOCHEMISTRY

FACULTY OF MEDICINE,HASANUDDIN UNIVERSITY,MAKASSAR


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DNA to Protein• DNA acts as a “manager” in the

process of making proteins• DNA is the template or startingsequence that is copied into RNAthat is then used to make theprotein


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Central Dogma

• One gene – one protein


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Central Dogma

• This is the same for bacteria tohumans

• DNA is the genetic instruction or gene• DNA RNA is called Transcription

– RNA chain is called a transcript

• RNA Protein is called Translation


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Expression of

Genes

• Some genes aretranscribed in

large quantitiesbecause we needlarge amount ofthis protein

• Some genes aretranscribed insmall quantities

because we needonly a smallamount of thisprotein


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Transcription

• Copy the gene of interest into RNAwhich is made up of nucleotides linkedby phosphodiester bonds – like DNA

• RNA differs from DNA– Ribose is the sugar rather thandeoxyribose – ribonucleotides

– U instead of T; A, G and C the same

– Single stranded• Can fold into a variety of shapes that allows

RNA to have structural and catalyticfunctions


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RNA Differences


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RNA Differences


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Transcription

• Similarities to DNA replication– Open and unwind a portion of the DNA– 1 strand of the DNA acts as a template

– Complementary base-pairing with DNA• Differences– RNA strand does not stay paired with

DNA

• DNA re-coils and RNA is single stranded– RNA is shorter than DNA

• RNA is several 1000 bp or shorter whereasDNA is 250 million bp long


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Template to Transcripts

• The RNA transcript is identical to theNON-template strand with the exceptionof the T‟s becoming U‟s


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RNA Polymerase• Catalyzes theformation of the

phosphodiester bondsbetween thenucleotides (sugar tophosphate)

• Uncoils the DNA, addsthe nucleotide one at atime in the 5‟ to 3‟fashion

• Uses the energytrapped in thenucleotides themselvesto form the new bonds


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RNA Elongation

• Reads template3‟ to 5‟

• Adds nucleotides

5‟ to 3‟ (5‟phosphate to 3‟hydroxyl)

• Synthesis is thesame as theleading strand ofDNA


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RNA Polymerase

• RNA is released so we can make manycopies of the gene, usually before the firstone is done– Can have multiple RNA polymerase molecules on

a gene at a time


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Differences in

DNA and RNA Polymerases• RNA polymerase adds ribonucleotides not

deoxynucleotides

• RNA polymerase does not have the ability toproofread /koreksi /what they transcribe

• RNA polymerase can work without a primer

• RNA will have an error 1 in every 10,000nucleotides (DNA is 1 in 10,000,000nucleotides)


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Types of RNA

• messenger RNA (mRNA) – codes forproteins

• ribosomal RNA (rRNA) – forms thecore of the ribosomes, machinery formaking proteins

• transfer RNA (tRNA) – carries theamino acid for the growing proteinchain


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DNA Transcription in Bacteria

• RNA polymerase must know wherethe start of a gene is in order to copy

it• RNA polymerase has weakinteractions with the DNA unless itencounters a promoter (kec ada)– A promoter is a specific sequence of

nucleotides that indicate the start sitefor RNA synthesis


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RNA Synthesis

• RNA pol opensthe DNA doublehelix and createsthe template

• RNA pol moves ntby nt, unwindsthe DNA as itgoes

• Will stop when it

encounters aSTOP codon,RNA pol leaves,releasing theRNA strand

Sigma factor


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Start and Stop Sequences


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Start dan stop sinyal


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DNA Transcribed

• The strand of DNA transcribed is dependent on which

strand the promoter is on• Once RNA polymerase is bound to promoter, no option

but to transcribe the appropriate DNA strand• Genes may be adjacent to one another or on opposite

strands


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Eukaryotic Transcription

• Transcription occurs in the nucleus in eukaryotes,nucleoid in bacteria• Translation occurs on ribosomes in the cytoplasm• mRNA is transported out of nucleus through the

nuclear pores


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RNA Processing

• Eukaryotic cells process the RNA in thenucleus before it is moved to thecytoplasm for protein synthesis

• The RNA that is the direct copy of theDNA is the primary transcript

• 2 methods used to process primary

transcripts to increase the stability ofmRNA being exported to the cytoplasm– RNA capping– Polyadenylation


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RNA Processing

• RNA capping happens at the 5‟ end of the RNA,usually adds a methylguanosine shortly after RNApolymerase makes the 5‟ end of the primarytranscript

• Polyadenylation modifies the 3‟ end of the primarytranscript by the addition of a string of A‟s


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C di d N di S


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Coding and Non-coding Sequences

• In bacteria, the RNA made is translated to a

protein• In eukaryotic cells, the primary transcript is madeof coding sequences called exons and non-codingsequences called introns

• It is the exons that make up the mRNA that gets

translated to a protein

RNA S li i


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RNA Splicing• Responsible for the removal of the introns to create

the mRNA• Introns contain sequences that act as cues for theirremoval

• Carried out by small nuclear riboprotein particles (snRNPs)


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snRNPs

• snRNPs cometogether and cut

out the intron andrejoin the ends ofthe RNA

• Intron is removed

as a lariat – loop ofRNA like a cowboyrope

B fit f S li i


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Benefits of Splicing

• Allows for genetic recombination

– Link exons from different genes together to createa new mRNA

• Also allows for 1 primary transcript to encodefor multiple proteins by rearrangement of the

exons


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Summary


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RNA to Protein

• Translation is the process ofturning mRNA into protein

• Translate from one “language”(mRNA nucleotides) to a second“language” (amino acids)

• Genetic code – nucleotidesequence that is translated toamino acids of the protein


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Translasi

• Tiga nukleotida mengkode satu asamamino

• Kode tidak tumpang tindih• Kode tidak ada jeda

• Kode genetik, 61 triplet untuk kode

asam amino dan 3 untuk kode stop

D DNA C d


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Degenerate DNA Code

• Nucleotides read 3 at a time meaning thatthere are 64 combinations for a codon (setof 3 nucleotides)

• Only 20 amino acids– More than 1 codon per AA – degenerate code

with the exception of Met and Trp (leastabundant AAs in proteins)


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Reading Frames

• Translation can occur in 1 of 3 possiblereading frames, dependent on wheredecoding starts in the mRNA


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Transfer RNAMolecules

• Translation requires anadaptor molecule that

recognizes the codonon mRNA and at adistant site carries theappropriate amino acid

• Intra-strand basepairing allows for thischaracteristic shape

• Anticodon is oppositefrom where the aminoacid is attached


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Wobble Base Pairing

• Due to degenerate code for amino acids some

tRNA can recognize several codons becausethe 3rd spot can wobble or be mismatched

• Allows for there only being 31 tRNA for the61 codons


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Attachment of AA to tRNA

• Aminoacyl-tRNA synthase is the

enzyme responsible for linking theamino acid to the tRNA

• A specific enzyme for each aminoacid and not for the tRNA


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2 „Adaptors‟ TranslateGenetic Code to Protein

1

2


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Ribosomes• Complex machinery that

controls protein synthesis

• 2 subunits– 1 large – catalyzes thepeptide bond formation

– 1 small – binds mRNA and

tRNA• Contains protein and RNA

– rRNA central to the catalyticactivity

• Folded structure is highlyconserved

– Protein has less homology andmay not be as important


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Ribosome Structures


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Ribosome Structures

• May be free in cytoplasm or attached to the ER

• Subunits made in the nucleus in the nucleolus and

transported to the cytoplasm

Rib l S b i


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Ribosomal Subunits

• 1 large subunit – catalyzes the formation of the peptidebond

• 1 small subunit – matches the tRNA to the mRNA• Moves along the mRNA adding amino acids to growing

protein chain

Ribosomal Movement


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Ribosomal Movement

• 4 binding sites– mRNA binding site

– Peptidyl-tRNA binding site (P-site)• Holds tRNA attached to growing end of the peptide

– Aminoacyl-tRNA binding site (A-site)• Holds the incoming AA

– Exit site (E-site)

E-site

El h


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3 Step Elongation Phase• Elongation is a cycle of events

• Step 1 – aminoacyl-tRNA comes intoempty A-site next to the occupied P-site; pairs with the codon

• Step 2 – C‟ end of peptide chain

uncouples from tRNA in P-site andlinks to AA in A-site– Peptidyl transferase responsible for bond

formation– Each AA added carries the energy for the

addition of the next AA• Step 3 – peptidyl-tRNA moves to theP-site; requires hydrolysis of GTP– tRNA released back to the cytoplasmic

pool

Initi ti n P ss


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Initiation Process

• Determines whether mRNA issynthesized and sets the readingframe that is used to make theprotein

• Initiation process brings the ribosomalsubunits together at the site wherethe peptide should begin

• Initiator tRNA brings in Met– Initiator tRNA is different than the

tRNA that adds other Met

Rib s l Ass bl


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Ribosomal AssemblyInitiation Phase

• Initiation factors (IFs) catalyze thesteps – not well defined

• Step 1 – small ribosomal subunit withthe IF finds the start codon –AUG– Moves 5‟ to 3‟ on mRNA

– Initiator tRNA brings in the 1st

AA whichis always Met and then can bind themRNA

• Step 2 – IF leaves and then largesubunit can bind – protein synthesis

continues• Met is at the start of every proteinuntil post-translational modificationtakes place

Eukaryotic vs Procaryotic


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Eukaryotic vs Procaryotic

• Procaryotic

– No CAP; have specific ribosome binding site upstream of AUG– Polycistronic – multiple proteins from same mRNA

• Eucaryotic– Monocistronic – one polypeptide per mRNA


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P t i R l


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Protein Release

• Protein released when a STOPcodon is encountered– UAG, UAA, UGA (must know these

sequences!)

• Cytoplasmic release factors bind to the stop codon thatgets to the A-site; alters thepeptidyl transferase and addsH

2

O instead of an AA• Protein released and the

ribosome breaks into the 2subunits to move on to anothermRNA


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Polyribosomes• As the ribosome

moves down the

mRNA, it allows forthe addition ofanother ribosomeand the start ofanother protein

• Each mRNA hasmultiple ribosomes

attached,polyribosome orpolysome


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Regulation of Protein Synthesis

• Lifespan of proteins vary, needmethod to remove old ordamaged proteins

• Enzymes that degrade

proteins are called proteases – process is called proteolysis

• In the cytosol there are largecomplexes of proteolyticenzymes that remove damagedproteins

• Ubiquitin, small protein, isadded as a tag for disposal ofprotein


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Protein Synthesis

• Protein synthesis takes the mostenergy input of all the biosynthetic

pathways• 4 high-energy bonds required foreach AA addition– 2 in charging the tRNA (adding AA)

– 2 in ribosomal activities (step 1 and step3 of elongation phase)


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Summary


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Ribozyme

• A RNA molecule can folddue to its single strandednature and in folding cancause the cleavage ofother RNA molecules

• A RNA molecule that

functions like an enzymehence ribozyme name

Ek i


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Ekspresi gen

DNA Transkripsi RNA Translasi Protein

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Chapter 7 - DNA to Protein_2

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