Chapter 7Chapter 7

From DNA to ProteinFrom DNA to Protein

DNA to ProteinDNA to ProteinDNA acts as a “manager” in the DNA acts as a “manager” in the

process of making proteinsprocess of making proteinsDNA is the template or starting DNA is the template or starting

sequence that is copied into RNA sequence that is copied into RNA that is then used to make the proteinthat is then used to make the protein

Central DogmaCentral Dogma

One gene – one proteinOne gene – one protein

Central DogmaCentral Dogma

This is the same for bacteria to humansThis is the same for bacteria to humansDNA is the genetic instruction or geneDNA is the genetic instruction or geneDNA DNA RNA is called RNA is called TranscriptionTranscription

RNA chain is called aRNA chain is called a transcripttranscriptRNA RNA Protein is called Protein is called TranslationTranslation

Expression of Expression of GenesGenes

Some genes are Some genes are transcribed in transcribed in large quantities large quantities because we need because we need large amount of large amount of this proteinthis protein

Some genes are Some genes are transcribed in transcribed in small quantities small quantities because we need because we need only a small only a small amount of this amount of this proteinprotein

TranscriptionTranscription

Copy the gene of interest into RNA Copy the gene of interest into RNA which is made up of nucleotides linked which is made up of nucleotides linked by phosphodiester bonds – like DNAby phosphodiester bonds – like DNA

RNA differs from DNARNA differs from DNARibose is the sugar rather than Ribose is the sugar rather than

deoxyribose – deoxyribose – ribonucleotidesribonucleotides U instead of T; A, G and C the sameU instead of T; A, G and C the sameSingle strandedSingle stranded

Can fold into a variety of shapes that allows Can fold into a variety of shapes that allows RNA to have structural and catalytic functionsRNA to have structural and catalytic functions

RNA RNA DifferencesDifferences

RNA DifferencesRNA Differences

TranscriptionTranscription

Similarities to DNA replicationSimilarities to DNA replicationOpen and unwind a portion of the DNAOpen and unwind a portion of the DNA1 strand of the DNA acts as a template1 strand of the DNA acts as a templateComplementary base-pairing with DNAComplementary base-pairing with DNA

Differences Differences RNA strand does not stay paired with DNARNA strand does not stay paired with DNA

DNA re-coils and RNA is single strandedDNA re-coils and RNA is single strandedRNA is shorter than DNARNA is shorter than DNA

RNA is several 1000 bp or shorter whereas RNA is several 1000 bp or shorter whereas DNA is 250 million bp longDNA is 250 million bp long

Template to TranscriptsTemplate to Transcripts

The RNA transcript is identical to the NON-The RNA transcript is identical to the NON-template strand with the exception of the T’s template strand with the exception of the T’s becoming U’sbecoming U’s

RNA RNA PolymerasePolymerase

Catalyzes the formation Catalyzes the formation of the phosphodiester of the phosphodiester bonds between the bonds between the nucleotides (sugar to nucleotides (sugar to phosphate)phosphate)

Uncoils the DNA, adds Uncoils the DNA, adds the nucleotide one at a the nucleotide one at a time in the 5’ to 3’ time in the 5’ to 3’ fashionfashion

Uses the energy Uses the energy trapped in the trapped in the nucleotides themselves nucleotides themselves to form the new bondsto form the new bonds

RNA ElongationRNA Elongation

Reads template 3’ Reads template 3’ to 5’to 5’

Adds nucleotides Adds nucleotides 5’ to 3’ (5’ 5’ to 3’ (5’ phosphate to 3’ phosphate to 3’ hydroxyl)hydroxyl)

Synthesis is the Synthesis is the same as the same as the leading strand of leading strand of DNADNA

RNA PolymeraseRNA Polymerase RNA is released so we can make many RNA is released so we can make many

copies of the gene, usually before the first copies of the gene, usually before the first one is doneone is done Can have multiple RNA polymerase molecules on Can have multiple RNA polymerase molecules on

a gene at a timea gene at a time

Differences in Differences in DNA and RNA PolymerasesDNA and RNA Polymerases

RNA polymerase adds ribonucleotides not RNA polymerase adds ribonucleotides not deoxynucleotidesdeoxynucleotides

RNA polymerase does not have the ability to RNA polymerase does not have the ability to proofreadproofread what they transcribe what they transcribe

RNA polymerase can work without a primerRNA polymerase can work without a primer RNA will have an error 1 in every 10,000 RNA will have an error 1 in every 10,000

nucleotides (DNA is 1 in 10,000,000 nucleotides (DNA is 1 in 10,000,000 nucleotides)nucleotides)

Types of RNATypes of RNA

messenger RNA (mRNA)messenger RNA (mRNA) – codes for – codes for proteinsproteins

ribosomal RNA (rRNA)ribosomal RNA (rRNA) – forms the core – forms the core of the ribosomes, machinery for making of the ribosomes, machinery for making proteinsproteins

transfer RNA (tRNA)transfer RNA (tRNA) – carries the amino – carries the amino acid for the growing protein chainacid for the growing protein chain

DNA Transcription in BacteriaDNA Transcription in Bacteria

RNA polymerase must know where the RNA polymerase must know where the start of a gene is in order to copy itstart of a gene is in order to copy it

RNA polymerase has weak interactions RNA polymerase has weak interactions with the DNA unless it encounters a with the DNA unless it encounters a promoterpromoterA promoter is a specific sequence of A promoter is a specific sequence of

nucleotides that indicate the start site for RNA nucleotides that indicate the start site for RNA synthesissynthesis

RNA SynthesisRNA Synthesis RNA pol opens RNA pol opens

the DNA double the DNA double helix and creates helix and creates the the templatetemplate

RNA pol moves nt RNA pol moves nt by nt, unwinds the by nt, unwinds the DNA as it goesDNA as it goes

Will stop when it Will stop when it encounters a encounters a STOP codon, STOP codon, RNA pol leaves, RNA pol leaves, releasing the RNA releasing the RNA strandstrand

Sigma (Sigma () Factor) Factor

Part of the bacterial RNA polymerase that Part of the bacterial RNA polymerase that helps it recognize the promoterhelps it recognize the promoter

Released after about 10 nucleotides of Released after about 10 nucleotides of RNA are linked togetherRNA are linked together

Rejoins with a released RNA polymerase Rejoins with a released RNA polymerase to look for a new promoterto look for a new promoter

Start and Stop SequencesStart and Stop Sequences

DNA TranscribedDNA Transcribed

The strand of DNA transcribed is dependent on which The strand of DNA transcribed is dependent on which strand the promoter is onstrand the promoter is on

Once RNA polymerase is bound to promoter, no option but Once RNA polymerase is bound to promoter, no option but to transcribe the appropriate DNA strandto transcribe the appropriate DNA strand

Genes may be adjacent to one another or on opposite Genes may be adjacent to one another or on opposite strandsstrands

Eukaryotic TranscriptionEukaryotic Transcription

Transcription occurs in the Transcription occurs in the nucleusnucleus in eukaryotes, in eukaryotes, nucleoidnucleoid in bacteria in bacteria

Translation occurs on ribosomes in the cytoplasmTranslation occurs on ribosomes in the cytoplasm mRNA is transported out of nucleus through the mRNA is transported out of nucleus through the

nuclear poresnuclear pores

RNA ProcessingRNA Processing

Eukaryotic cells process the RNA in the Eukaryotic cells process the RNA in the nucleus before it is moved to the nucleus before it is moved to the cytoplasm for protein synthesiscytoplasm for protein synthesis

The RNA that is the direct copy of the The RNA that is the direct copy of the DNA is the DNA is the primary transcriptprimary transcript

2 methods used to process primary 2 methods used to process primary transcripts to increase the stability of transcripts to increase the stability of mRNA being exported to the cytoplasmmRNA being exported to the cytoplasmRNA cappingRNA cappingPolyadenylationPolyadenylation

RNA ProcessingRNA Processing

RNA cappingRNA capping happens at the 5’ end of the RNA, happens at the 5’ end of the RNA, usually adds a methylgaunosine shortly after RNA usually adds a methylgaunosine shortly after RNA polymerase makes the 5’ end of the primary polymerase makes the 5’ end of the primary transcripttranscript

PolyadenylationPolyadenylation modifies the 3’ end of the primary modifies the 3’ end of the primary transcript by the addition of a string of A’stranscript by the addition of a string of A’s

Coding and Non-coding SequencesCoding and Non-coding Sequences

In bacteria, the RNA made is translated to a proteinIn bacteria, the RNA made is translated to a protein In eukaryotic cells, the primary transcript is made of In eukaryotic cells, the primary transcript is made of

coding sequences called coding sequences called exonsexons and non-coding and non-coding sequences called sequences called intronsintrons

It is the exons that make up the mRNA that gets It is the exons that make up the mRNA that gets translated to a proteintranslated to a protein

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

mRNAmRNA Introns contain sequences that act as cues for their Introns contain sequences that act as cues for their

removalremoval Carried out by Carried out by small nuclear riboprotein particlessmall nuclear riboprotein particles

(snRNPs)(snRNPs)

snRNPssnRNPs

snRNPs come snRNPs come together and cut out together and cut out the intron and rejoin the intron and rejoin the ends of the RNAthe ends of the RNA

Intron is removed as Intron is removed as a lariat – loop of a lariat – loop of RNA like a cowboy RNA like a cowboy roperope

Benefits of SplicingBenefits of Splicing Allows for genetic recombinationAllows for genetic recombination

Link exons from different genes together to create a Link exons from different genes together to create a new mRNAnew mRNA

Also allows for 1 primary transcript to encode for Also allows for 1 primary transcript to encode for multiple proteins by rearrangement of the exonsmultiple proteins by rearrangement of the exons

SummarySummary

RNA to ProteinRNA to Protein

Translation is the process of Translation is the process of turning mRNA into proteinturning mRNA into protein

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

Genetic codeGenetic code – nucleotide – nucleotide sequence that is translated to sequence that is translated to amino acids of the proteinamino acids of the protein

Degenerate DNA CodeDegenerate DNA Code

Nucleotides read 3 at a time meaning that Nucleotides read 3 at a time meaning that there are 64 combinations for a there are 64 combinations for a codoncodon (set of (set of 3 nucleotides)3 nucleotides)

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

the exception of Met and Trp (least abundant AAs the exception of Met and Trp (least abundant AAs in proteins)in proteins)

Reading Reading FramesFrames

Translation can occur in 1 of 3 possible reading Translation can occur in 1 of 3 possible reading frames, dependent on where decoding starts in frames, dependent on where decoding starts in the mRNAthe mRNA

Transfer RNA Transfer RNA MoleculesMolecules

Translation requires an Translation requires an adaptoradaptor molecule that molecule that recognizes the codon on recognizes the codon on mRNA and at a distant mRNA and at a distant site carries the site carries the appropriate amino acidappropriate amino acid

Intra-strand base pairing Intra-strand base pairing allows for this allows for this characteristic shapecharacteristic shape

AnticodonAnticodon is opposite is opposite from where the amino from where the amino acid is attachedacid is attached

Wobble Base Wobble Base PairingPairing

Due to degenerate code for amino acids some Due to degenerate code for amino acids some tRNA can recognize several codons because tRNA can recognize several codons because the 3the 3rdrd spot can wobble or be mismatched spot can wobble or be mismatched

Allows for there only being 31 tRNA for the 61 Allows for there only being 31 tRNA for the 61 codonscodons

Attachment of AA to tRNAAttachment of AA to tRNA

Aminoacyl-tRNA synthaseAminoacyl-tRNA synthase is the is the enzyme responsible for linking the enzyme responsible for linking the amino acid to the tRNAamino acid to the tRNA

A specific enzyme for each amino acid A specific enzyme for each amino acid and not for the tRNAand not for the tRNA

2 ‘Adaptors’ Translate 2 ‘Adaptors’ Translate Genetic Code to ProteinGenetic Code to Protein

1

2

RibosomesRibosomes Complex machinery that Complex machinery that

controls protein synthesiscontrols protein synthesis 2 subunits2 subunits

1 large – catalyzes the peptide 1 large – catalyzes the peptide bond formationbond formation

1 small – binds mRNA and 1 small – binds mRNA and tRNAtRNA

Contains protein and RNAContains protein and RNA rRNA central to the catalytic rRNA central to the catalytic

activityactivity Folded structure is highly Folded structure is highly

conserved conserved

Protein has less homology and Protein has less homology and may not be as importantmay not be as important

Ribosome StructuresRibosome Structures

May be free in cytoplasm or attached to the ERMay be free in cytoplasm or attached to the ER Subunits made in the nucleus in the nucleolus and Subunits made in the nucleus in the nucleolus and

transported to the cytoplasmtransported to the cytoplasm

Ribosomal SubunitsRibosomal Subunits

1 large subunit – catalyzes the formation of the peptide bond1 large subunit – catalyzes the formation of the peptide bond 1 small subunit – matches the tRNA to the mRNA1 small subunit – matches the tRNA to the mRNA Moves along the mRNA adding amino acids to growing Moves along the mRNA adding amino acids to growing

protein chainprotein chain

Ribosomal MovementRibosomal Movement

4 binding sites4 binding sites mRNA binding sitemRNA binding site Peptidyl-tRNA binding site (P-site)Peptidyl-tRNA binding site (P-site)

Holds tRNA attached to growing end of the peptideHolds tRNA attached to growing end of the peptide

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

Exit site (E-site)Exit site (E-site)

E-site

3 Step Elongation 3 Step Elongation Phase Phase

Elongation is a cycle of eventsElongation is a cycle of events Step 1 – aminoacyl-tRNA comes into Step 1 – aminoacyl-tRNA comes into

empty A-site next to the occupied P-site; empty A-site next to the occupied P-site; pairs with the codonpairs with the codon

Step 2 – C’ end of peptide chain Step 2 – C’ end of peptide chain uncouples from tRNA in P-site and links uncouples from tRNA in P-site and links to AA in A-siteto AA in A-site Peptidyl transferasePeptidyl transferase responsible for bond responsible for bond

formationformation Each AA added carries the energy for the Each AA added carries the energy for the

addition of the next AAaddition of the next AA Step 3 – peptidyl-tRNA moves to the P-Step 3 – peptidyl-tRNA moves to the P-

site; requires hydrolysis of GTPsite; requires hydrolysis of GTP tRNA released back to the cytoplasmic pooltRNA released back to the cytoplasmic pool

Initiation ProcessInitiation ProcessDetermines whether mRNA is Determines whether mRNA is

synthesized and sets the reading frame synthesized and sets the reading frame that is used to make the protein that is used to make the protein

Initiation process brings the ribosomal Initiation process brings the ribosomal subunits together at the site where the subunits together at the site where the peptide should begin peptide should begin

Initiator tRNAInitiator tRNA brings in Met brings in Met Initiator tRNA is different than the tRNA that Initiator tRNA is different than the tRNA that

adds other Metadds other Met

Ribosomal Assembly Ribosomal Assembly Initiation Phase Initiation Phase

Initiation factors (IFs) catalyze the steps Initiation factors (IFs) catalyze the steps – not well defined– not well defined

Step 1 – small ribosomal subunit with Step 1 – small ribosomal subunit with the IF finds the start codon –AUG the IF finds the start codon –AUG Moves 5’ to 3’ on mRNAMoves 5’ to 3’ on mRNA Initiator tRNA brings in the 1Initiator tRNA brings in the 1stst AA which is AA which is

always Met and then can bind the mRNAalways Met and then can bind the mRNA Step 2 – IF leaves and then large Step 2 – IF leaves and then large

subunit can bind – protein synthesis subunit can bind – protein synthesis continuescontinues

Met is at the start of every protein until Met is at the start of every protein until post-translational modification takes post-translational modification takes placeplace

Eukaryotic vs ProcaryoticEukaryotic vs Procaryotic

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

Eucaryotic Eucaryotic MonocistronicMonocistronic – one polypeptide per mRNA – one polypeptide per mRNA

Protein ReleaseProtein Release Protein released when a STOP Protein released when a STOP

codon is encounteredcodon is encountered UAG, UAA, UGA (must know these UAG, UAA, UGA (must know these

sequences!)sequences!) Cytoplasmic Cytoplasmic release factorsrelease factors bind bind

to the stop codon that gets to the to the stop codon that gets to the A-site; alters the peptidyl A-site; alters the peptidyl transferase and adds Htransferase and adds H22O instead O instead of an AAof an AA

Protein released and the Protein released and the ribosome breaks into the 2 ribosome breaks into the 2 subunits to move on to another subunits to move on to another mRNAmRNA

PolyribosomesPolyribosomes As the ribosome As the ribosome moves down the moves down the mRNA, it allows for mRNA, it allows for the addition of the addition of another ribosome another ribosome and the start of and the start of another proteinanother protein

Each mRNA has Each mRNA has multiple ribosomes multiple ribosomes attached, attached, polyribosome or polyribosome or polysomepolysome

Regulation of Protein SynthesisRegulation of Protein Synthesis

Lifespan of proteins vary, need Lifespan of proteins vary, need method to remove old or method to remove old or damaged proteinsdamaged proteins

Enzymes that degrade proteins Enzymes that degrade proteins are called are called proteasesproteases – process – process is called is called proteolysisproteolysis

In the cytosol there are large In the cytosol there are large complexes of proteolytic complexes of proteolytic enzymes that remove damaged enzymes that remove damaged proteinsproteins

Ubiquitin, small protein, is added Ubiquitin, small protein, is added as a tag for disposal of proteinas a tag for disposal of protein

Protein SynthesisProtein Synthesis

Protein synthesis takes the most energy Protein synthesis takes the most energy input of all the biosynthetic pathwaysinput of all the biosynthetic pathways

4 high-energy bonds required for each 4 high-energy bonds required for each AA additionAA addition2 in 2 in chargingcharging the tRNA (adding AA) the tRNA (adding AA)2 in ribosomal activities (step 1 and step 3 2 in ribosomal activities (step 1 and step 3

of elongation phase)of elongation phase)

SummarySummary

RibozymeRibozyme

A RNA molecule can fold A RNA molecule can fold due to its single stranded due to its single stranded nature and in folding can nature and in folding can cause the cleavage of other cause the cleavage of other RNA moleculesRNA molecules

A RNA molecule that A RNA molecule that functions like an enzyme functions like an enzyme hence hence ribozymeribozyme name name