Genetic code, transcription & Genetic code, transcription & translationtranslation

I.I. Overview: central dogmaOverview: central dogma

II.II. The genetic codeThe genetic code

A.A. Features of the Genetic CodeFeatures of the Genetic Code

B.B. The reading frameThe reading frame

III.III. Going from gene to protein, part one, Going from gene to protein, part one, TranscriptionTranscription

A.A. Properties of RNAProperties of RNA

B.B. Classes of RNAClasses of RNA

C.C. RNA polymeraseRNA polymerase

D.D. Initiation, Elongation & TerminationInitiation, Elongation & Termination

E.E. Transcription in EukaryotesTranscription in Eukaryotes

IV.IV. TranslationTranslation

I. Overview: Flow of genetic information I. Overview: Flow of genetic information encoded in DNA to mRNA to proteinencoded in DNA to mRNA to protein

II. The Genetic Code:64 codons in all, 1 start codon, 3 stop codons, 61 code for

amino acids• Collection of codons that corresponds to each amino acid

A.A. Features of the Genetic Code:Features of the Genetic Code:1.1. Written in linear form, mRNA derived from the Written in linear form, mRNA derived from the

complementary nucleotide bases in DNAcomplementary nucleotide bases in DNA2.2. 4 letters from which to choose @ each position, a 3 letter 4 letters from which to choose @ each position, a 3 letter

codon would make 64 words. codon would make 64 words. TripletTriplet (4 (433 = 64 possible = 64 possible combinations), combinations),

• Determined by Nirenberg & Matthei (1961), Nirenberg & Leder Determined by Nirenberg & Matthei (1961), Nirenberg & Leder (1964), UUU UUU UUU UUU = phe-phe-phe(1964), UUU UUU UUU UUU = phe-phe-phe

3.3. UnambiguousUnambiguous, each triplet specifies only 1 amino acid., each triplet specifies only 1 amino acid.4.4. RedundantRedundant, , degenerate degenerate (a given amino acid can be

specified by more than one codon)5.5. Contains “start” Contains “start” (AUG)(AUG) and “stop” (UGA, UAA, UAG) signals and “stop” (UGA, UAA, UAG) signals6.6. No “commas” used, codons read one after another without No “commas” used, codons read one after another without

breaksbreaks7.7. Non-overlappingNon-overlapping

• AACAACAACAAC = Asn, Thr, GlnAACAACAACAAC = Asn, Thr, Gln

8.8. ““Nearly Universal” – all life shares the same code, w/a few Nearly Universal” – all life shares the same code, w/a few exceptions:exceptions:

• Mitochondrial DNA, Fungi & Algae Mitochondrial DNA, Fungi & Algae

B. The Reading Frame: gene read in one direction, if each 3 bases makes a word then the reading frame is established by taking the first 3 bases from the end as the first word….

Transcription

Enzymatic synthesis of RNA, using 1 of the DNA strands for a particular gene as the template.

III. How do we get from gene to polypeptide?Going from gene to protein, part one- (transcription):

Occurs in the nucleus in eukaryotes – so separate from translationIn Prokaryotes, it is coupled with translation (no nucleus)Requires chromatin remodelingInvolves interaction between upstream DNA sequences & protein factorsInvolves processing

A. Properties of RNAA. Properties of RNA

1)1) Single strandedSingle stranded

2)2) Highly Highly flexibleflexible

3)3) Ribose sugarRibose sugar

4)4) Uracil instead of Uracil instead of ThymineThymine

5)5) Can catalyze Can catalyze biological rxnsbiological rxns

Classes of RNAsClasses of RNAsInformational RNAs = mRNAInformational RNAs = mRNA

Prokaryotes vs. eukaryotesProkaryotes vs. eukaryotes

Functional RNAs = tRNA, rRNA, snRNA, miRNA, siRNAFunctional RNAs = tRNA, rRNA, snRNA, miRNA, siRNA

Initiation involves the Binding of Initiation involves the Binding of RNA PolymeraseRNA Polymerase

PromoterPromoter = specific sequences of DNA = specific sequences of DNA where the RNA polymerase bindswhere the RNA polymerase binds

The Promoter region is The Promoter region is recognized by the recognized by the sigma subunitsigma subunit of the RNA polymerase of the RNA polymerase

The Enzyme “explores” DNA until it The Enzyme “explores” DNA until it recognizes the promoter region & recognizes the promoter region & bindsbinds to to 60 bp of the helix60 bp of the helix

Promoters – consensus sequencesPromoters – consensus sequences

In different In different E. Coli E. Coli strainsstrains

-35 -10 +1

Mutations in the promoter region can have serious effects:down mutations – make the promoter “weaker”up mutations – make the promoter “stronger”Insertions/deletions that change the base # between -10 & -35 region are deleterious.

RNA polymeraseRNA polymerase5 subunits:5 subunits:

Core enzyme (β & β’ provide active site Core enzyme (β & β’ provide active site for transcription)for transcription)

Sigma factor – involved in initiationSigma factor – involved in initiation

HoloenzymeHoloenzyme

Initiation & ElongationInitiation & Elongation

1)1) Initial loose binding of holoenzymeInitial loose binding of holoenzyme

2)2) Formation of the Open promoter complexFormation of the Open promoter complex SigmaSigma binds tightly to promoter and then locally melts DNA binds tightly to promoter and then locally melts DNA ds DNA becomes temporarily strand separated to allow ds DNA becomes temporarily strand separated to allow one one

strandstrand to be used as template to be used as template

3)3) Elongation -Each new rNTP is added one by one, chosen by its Elongation -Each new rNTP is added one by one, chosen by its ability to base pair with the DNA template (5’ to 3’ direction)ability to base pair with the DNA template (5’ to 3’ direction)

Nucleosides

…

…

3’

3’

5’

Mechanism of chain elongation, catalyzed by RNA polymerase.

Energy for the reaction is derived from splitting the triphosphate, and releasing the inorganic diphospates.

Termination, final stageTermination, final stageTerminator sequence – GC rich stretch Terminator sequence – GC rich stretch followed by several A’s on the template followed by several A’s on the template strandstrand

1) Rho independent/Intrinsic terminator1) Rho independent/Intrinsic terminator

2). Rho dependent 2). Rho dependent terminationtermination

An additional protein factor is needed for the dissociation of the RNA polymerase. The rho binds to the mRNA transcript and causes the RNA pol. to “stutter” and fall off.

Transcription in eukaryotes is Transcription in eukaryotes is different from prokaryotesdifferent from prokaryotes

ProkaryotesProkaryotes eukaryoteseukaryotes

Where it Where it happenshappens

Cytoplasm, coupled Cytoplasm, coupled w/translationw/translation

Nucleus, separate from Nucleus, separate from translationtranslation

Types of RNA Types of RNA polpol

Only oneOnly one 3 types3 types

RegulationRegulation promoterpromoter More complex -More complex -Promoter, enhancer, Promoter, enhancer, silencersilencer

ProcessingProcessing nonenone 5’ cap, 3’ tail, splicing, 5’ cap, 3’ tail, splicing, editingediting

Capping, polyadenylation & Capping, polyadenylation & SplicingSplicing

Capping of the 5’ end with methyl-guanosineCapping of the 5’ end with methyl-guanosine Protect, Enhance splicing efficiency, Enhance Protect, Enhance splicing efficiency, Enhance

translatability, Enhance transporttranslatability, Enhance transport

Addition of poly (A) tailAddition of poly (A) tail Protect & aid in translationProtect & aid in translation

SplicingSplicing Primary transcript – mRNA precursor that contains introns Primary transcript – mRNA precursor that contains introns mRNA maturation = introns removed through splicing to mRNA maturation = introns removed through splicing to

yield mature mRNA – snRNPs & snRNAs involvedyield mature mRNA – snRNPs & snRNAs involved Based upon signals that occur in the primary transcript:Based upon signals that occur in the primary transcript:

Exon/GU – intron – AG/ExonExon/GU – intron – AG/Exon

Wild type: met-trp-tyr-arg-gly-ser-pro-thrWild type: met-trp-tyr-arg-gly-ser-pro-thr

Mutant 1: met-trpMutant 1: met-trp

Mutant 2: met-trp-his-arg-gly-ser-pro-thrMutant 2: met-trp-his-arg-gly-ser-pro-thr

Mutant 3: met-cys-ile-val-val-gln-hisMutant 3: met-cys-ile-val-val-gln-his

2) The Pribnow box (or -10 region), TATA box, CAAT box and -35 region are 2) The Pribnow box (or -10 region), TATA box, CAAT box and -35 region are known as consensus sequences. What does this mean, and why are these known as consensus sequences. What does this mean, and why are these sequences important?sequences important?

1) Using the code, What type of 1) Using the code, What type of mutation occurred for each of mutation occurred for each of the 3 mutants?the 3 mutants?

Translation – getting from gene to Translation – getting from gene to polypeptide…polypeptide…

TranslationTranslation mRNA sequence is translated mRNA sequence is translated

into the polypeptide sequence into the polypeptide sequence at the ribosomeat the ribosome

1)1) Initiation: Ribosomes, Initiation: Ribosomes, rRNAs, tRNA & mRNA rRNAs, tRNA & mRNA assemble assemble

2)2) Elongation: mRNA Elongation: mRNA transcript is “read” at the transcript is “read” at the ribosome, tRNA brings the ribosome, tRNA brings the proper amino acids – one proper amino acids – one by oneby one

3)3) Termination: stop codons Termination: stop codons are not recognized by a are not recognized by a tRNA, but instead by tRNA, but instead by release factorsrelease factors

Functional RNAs involved in translationFunctional RNAs involved in translation

rRNA – ribosomal RNA, major rRNA – ribosomal RNA, major components of ribosomes (~80% of components of ribosomes (~80% of all RNA )all RNA )

tRNA – transfers amino acids to the tRNA – transfers amino acids to the ribosomeribosome Specific amino acid attaches to the 3’ Specific amino acid attaches to the 3’

end (dependent upon the end (dependent upon the anticodon anticodon sequencesequence))

Anticodon – binds to codon on mRNAAnticodon – binds to codon on mRNA ““charged tRNA”charged tRNA” – has the attached – has the attached

amino acid corresponding to its amino acid corresponding to its anticodonanticodon

Codon translation by Codon translation by tRNAtRNA Cloverleaf structure Cloverleaf structure

with two significant with two significant sites: anticodon loop sites: anticodon loop 3’ & the amino acid 3’ & the amino acid attachment site attachment site

AnticodonAnticodon in tRNA (3’- in tRNA (3’-5’) binds to the codon 5’) binds to the codon in mRNA (5’-3’)in mRNA (5’-3’)

Amino acids attached Amino acids attached to 3’ end via to 3’ end via aminoacyl-tRNA aminoacyl-tRNA sythetase = “charged”sythetase = “charged”

Codon-Anticodon Interactions

Polarity

5’ 3’3’ 5’

Codon on mRNA

Anticodon on tRNA

Anticodon loop

3’ 5’

C G I

(C, U)G C AmRNA 5’ 3’ mRNAs are always

read 5’ to 3’.

mRNAs are always read 5’ to 3’.

Ribosome structureRibosome structure

2 subunits, 50S (large) & 2 subunits, 50S (large) & 30S (small)30S (small)

The two subunits come The two subunits come together, bringing in the together, bringing in the tRNA & mRNA molecules tRNA & mRNA molecules to translate the mRNAto translate the mRNA

Has 3 binding sites for Has 3 binding sites for tRNA molecules, each tRNA molecules, each bound tRNA bridges the bound tRNA bridges the 30S and 50S subunits30S and 50S subunits

A siteA site P siteP site E siteE site

Initiation of TranslationInitiation of TranslationA.A. Formation of the Formation of the Pre-InitiationPre-Initiation complex: complex:

Polypeptide synthesis begins by the association Polypeptide synthesis begins by the association of the small subunit, mRNA and f-met-tRNAi of the small subunit, mRNA and f-met-tRNAi

Prokaryotes – Prokaryotes – Shine Dalgarno sequenceShine Dalgarno sequence Eukaryotes – binding to the Eukaryotes – binding to the 5’Cap5’Cap

B. Assembly of the Initiation FactorsB. Assembly of the Initiation FactorsIF = Initiation FactorIF = Initiation Factor, , Helps to establish the Helps to establish the initiation complexinitiation complex……In Prokaryotes: 1 IF Required for 30S subunit to bind with mRNA, In Prokaryotes: 1 IF Required for 30S subunit to bind with mRNA,

(IF-1 associates with the 30S ribosomal subunit in the A site and prevents (IF-1 associates with the 30S ribosomal subunit in the A site and prevents aminoacyl-tRNA from entering) aminoacyl-tRNA from entering)

Another IF helps tRNA to the ribosomeAnother IF helps tRNA to the ribosome (IF-2 binds to an initator tRNA and controls the entry of that tRNA into the (IF-2 binds to an initator tRNA and controls the entry of that tRNA into the

ribosome) ribosome) After associating with the 30S subunit, Charged initiator tRNA (f-met) binds After associating with the 30S subunit, Charged initiator tRNA (f-met) binds

to the IF-2 & IF-2 transfers the tRNA to the partial P site. When the 50S to the IF-2 & IF-2 transfers the tRNA to the partial P site. When the 50S subunit joins, a conformational change in the IF-2 causes IF-2 to release subunit joins, a conformational change in the IF-2 causes IF-2 to release and allow the 70S subunit to form and allow the 70S subunit to form

A third IF (IF-3) helps to stabilize thingsA third IF (IF-3) helps to stabilize thingsIn Eukaryotes: eIF’s involved in the formation of initation complexes In Eukaryotes: eIF’s involved in the formation of initation complexes with 5' mRNA and complexing & binding with Met-tRNAi, scanning with 5' mRNA and complexing & binding with Met-tRNAi, scanning mRNA for the initiator codon AUG, locating the binding site of initator mRNA for the initiator codon AUG, locating the binding site of initator tRNA to the AUG start site, and joining of the 60S subunit to create the tRNA to the AUG start site, and joining of the 60S subunit to create the 80S subunit 80S subunit

IFs bring initiation complex together

Charged fMet tRNA anticodon binds with AUG codon

Reading frame is set

Initiation complex complete, which then combines w/ large subunit

ElongationElongationA.A. Binding sites for two Binding sites for two

charged tRNA charged tRNA molecules are formed molecules are formed

P site (peptidyl)P site (peptidyl) A site (aminoacyl)A site (aminoacyl)

EF’s (elongation factors) mediate the entry EF’s (elongation factors) mediate the entry of charged tRNA’s into the A site.of charged tRNA’s into the A site.

The polypeptide chain on the peptidyl-tRNA The polypeptide chain on the peptidyl-tRNA is transferred to the charged tRNA, is transferred to the charged tRNA, Peptidyl transferase catalyzes the formation of Peptidyl transferase catalyzes the formation of

the peptide bond that links the two amino acids the peptide bond that links the two amino acids togethertogether

Translocation & continuationTranslocation & continuation

the ribosome then translocates to the next the ribosome then translocates to the next codon on the mRNA.codon on the mRNA.

Uncharged t-RNA released, newly formed Uncharged t-RNA released, newly formed peptidyl-tRNA moves from A site to P site.peptidyl-tRNA moves from A site to P site.

TerminationTermination

Stop codon: UAA, UAG, or UGAWhen stop codon reached, no charged tRNA exists to fill the A site, so elongation stopsNonsense supressor Mutations - alter the anticodon loops of specific tRNAs so they cannot recognize a stop codon in mRNA – an AA inserted in response to the stop codon, translation continues

Release factors – release the protein from the tRNA on the P site

Wobble HypothesisWobble Hypothesis

Degeneracy revisited - - - There are 64 Degeneracy revisited - - - There are 64 codons, but only 40-50 tRNAs?codons, but only 40-50 tRNAs?

Wobble hypothesis:Wobble hypothesis: the pairing between codon the pairing between codon and anticodon at the 1st two codon positions and anticodon at the 1st two codon positions always follows the usual rules, but “wobbles” always follows the usual rules, but “wobbles” occur at the 3rd position.occur at the 3rd position.-Reduces the number of tRNAs required-Reduces the number of tRNAs required-accounts for the observed degeneracy of -accounts for the observed degeneracy of the codethe code

Crick proposed that non-standard base-pairing might occur between the Anticodon base in 5’ position and the codon base in the 3’ position.

One gene: One enzyme hypothesisOne gene: One enzyme hypothesis

Beadle & Tatum investigated mutations in Beadle & Tatum investigated mutations in NeurosporaNeurospora

hypothesishypothesis: there was a one-to-one relationship between : there was a one-to-one relationship between genes and specific enzymesgenes and specific enzymes

Test: create genetic mutants that are unable to carry out specific Test: create genetic mutants that are unable to carry out specific enzymatic reactions enzymatic reactions

Used X-rays to create mutants & grew spores on enriched Used X-rays to create mutants & grew spores on enriched medium (mutants would survive)medium (mutants would survive)

Cultures transferred to minimal medium to check for mutantsCultures transferred to minimal medium to check for mutants

Genetic analysis showed that each mutant differed from the Genetic analysis showed that each mutant differed from the original, normal type by only one gene. original, normal type by only one gene.