Download - 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