Chapter 22 - Protein Chapter 22 - Protein SynthesisSynthesis

The ribosome, a The ribosome, a complex of RNA complex of RNA and protein, is the and protein, is the site where genetic site where genetic information is information is translated into translated into proteinprotein

mRNAmRNA

proteinproteinExam: Tues May 4 Exam: Tues May 4 12:00-3:0012:00-3:00

The Genetic CodeThe Genetic Code

• CodonsCodons - three letter genetic code - three letter genetic code (nonoverlapping)(nonoverlapping)

• tRNAtRNA - adapters between mRNA and proteins - adapters between mRNA and proteins

• Reading frameReading frame - each potential starting point for - each potential starting point for interpreting the 3 letter codeinterpreting the 3 letter code

•DNA – only four bases (A,T,G,C)DNA – only four bases (A,T,G,C)•Must code for 20 amino acidsMust code for 20 amino acids

•Two-base code: 4Two-base code: 422 = 16 combinations = 16 combinations•Four-base code: 4Four-base code: 444 = 256 combinations = 256 combinations•Three-base code: 4Three-base code: 433 = 64 combinations = 64 combinations

How is the info translated from NA to protein ?How is the info translated from NA to protein ?

Overlapping vs nonoverlapping reading of the Overlapping vs nonoverlapping reading of the three-letter codethree-letter code

What is one benefit ?What is one benefit ?

Three reading frames of mRNAThree reading frames of mRNA

•Translation of the correct message Translation of the correct message requires selection of the correct requires selection of the correct reading frame reading frame

tRNA ‘reads’ each codon Key: decide where to start

Standard genetic codeStandard genetic code

(5’-UU(5’-UUCC-3’)-3’)(5’-UU(5’-UUUU-3’)-3’)

(1962 – 1965, Khorana, Nirenberg in separate laboratories)(1962 – 1965, Khorana, Nirenberg in separate laboratories)

(START)(START)

Features of the genetic codeFeatures of the genetic code1.1. The genetic code is The genetic code is unambiguousunambiguous. Each codon . Each codon

corresponds to only corresponds to only one amino acid.one amino acid.

2. There are 2. There are multiple codons multiple codons for most amino acidsfor most amino acids(code is (code is degeneratedegenerate))

4. Codons with similar sequences specify similar amino 4. Codons with similar sequences specify similar amino acidsacids

5. Only 61 of the 64 codons specify amino acids5. Only 61 of the 64 codons specify amino acids

• TerminationTermination ( (stop codonsstop codons): ): UAA, UGA, UAGUAA, UGA, UAG

Initiation codonInitiation codon - Methionine codon ( - Methionine codon (AUGAUG) specifies initiation ) specifies initiation site for protein synthesissite for protein synthesis

3. The 3. The first two nucleotidesfirst two nucleotides of a codon are often enough of a codon are often enough

to specify a given amino acid (Gly = GG_ )to specify a given amino acid (Gly = GG_ )

Asp and Glu = GU_ single mutation chem similar AAAsp and Glu = GU_ single mutation chem similar AA

Crack the genetic codeCrack the genetic codeRNA Tie ClubRNA Tie Club

20 members20 members16 had a specific amino acid 16 had a specific amino acid 4 members had specific 4 members had specific

nucleotidenucleotide

George Gamow (ALA)George Gamow (ALA)F. Crick (TYR)F. Crick (TYR)

J. Watson (PRO)J. Watson (PRO)

Richard Feynman (GLY)Richard Feynman (GLY)

Melvin Calvin (HIS)Melvin Calvin (HIS)

Edward Teller (LEU)Edward Teller (LEU)

Leslie Orgel (THR)Leslie Orgel (THR)

Max Delbruck (TRY)Max Delbruck (TRY)

Sydney Brenner (VAL)Sydney Brenner (VAL)

Erwin Chargaff (LYS)Erwin Chargaff (LYS)

Combination of hard drinking and scienceCombination of hard drinking and science

11stst meeting meeting

Woods Hole 1954Woods Hole 1954

Crack the genetic codeCrack the genetic codeRNA Tie ClubRNA Tie Club

Aim: “Solve the riddle of RNA Aim: “Solve the riddle of RNA structure and understand structure and understand how it built proteins”how it built proteins”

George Gamow (ALA)George Gamow (ALA)

F. Crick (TYR)F. Crick (TYR)

J. Watson (PRO)J. Watson (PRO)

Richard Feynman (GLY)Richard Feynman (GLY)

Melvin Calvin (HIS)Melvin Calvin (HIS)

Edward Teller (LEU)Edward Teller (LEU)

Leslie Orgel (THR)Leslie Orgel (THR)

Max Delbruck (TRY)Max Delbruck (TRY)

Sydney Brenner (VAL)Sydney Brenner (VAL)

Erwin Chargaff (LYS)Erwin Chargaff (LYS)

Combination of hard drinking and scienceCombination of hard drinking and science

11stst meeting meeting

Woods Hole 1954Woods Hole 1954

Crack the genetic codeCrack the genetic code

George Gamow (ALA)George Gamow (ALA)

F. Crick (TYR)F. Crick (TYR)

““Adapter hypothesis”Adapter hypothesis”

Mathematics used to establish thatMathematics used to establish that

3 letter code would be enough to3 letter code would be enough todefine all 20 amino acidsdefine all 20 amino acids

Crack the genetic codeCrack the genetic codeNon–members of the RNA Tie Non–members of the RNA Tie

ClubClubMarshall NirenbergMarshall NirenbergJohann MatthaeiJohann Matthaei

‘‘Cell free system’Cell free system’RNA template, RNA template,

ribosomes,ribosomes,nucleotides, amino acids, nucleotides, amino acids, ATPATP

UUUUUUUUUUUUUUUUUUUUUUUU FFFFFFFF

CCCCCCCCCCCCCCCCCCCCCCCCCCCC PPPPPPPP

Har Gobind KhoranaHar Gobind Khorana

UCUCUCUCUUCUCUCUCU Ser,Leu,Ser,LeuSer,Leu,Ser,Leu

Robert HolleyRobert Holley

Determined the structure of Determined the structure of tRNAtRNA

Nobel prize Nobel prize 19681968

19611961

19651965

• Cloverleaf Cloverleaf structure of structure of tRNAtRNA

• Every cell must Every cell must contain at least 20 contain at least 20 tRNA (one for every tRNA (one for every amino acid)amino acid)

• Each tRNA must Each tRNA must recognize at least one recognize at least one codoncodon

Amino acidAmino acid

ComplementComplementary to the ary to the codoncodon

• Watson-Crick base Watson-Crick base pairing (dashed pairing (dashed lines)lines)

• tRNA has an tRNA has an acceptor stem and acceptor stem and four armsfour arms

• Conserved bases Conserved bases (gray)(gray)

However, the However, the 5’ anticodon5’ anticodon position position has some has some flexibilityflexibility in base pairing in base pairing

(the (the “wobble”“wobble” position) position)

• tRNA molecules are named for tRNA molecules are named for the amino acid that they carry the amino acid that they carry (e.g. (e.g. tRNAtRNAPhePhe) )

tRNA Anticodons Base-Pair with mRNA CodonstRNA Anticodons Base-Pair with mRNA Codons

• Base pairing between codon Base pairing between codon and anticodon is governed by and anticodon is governed by rules of Watson-Crick rules of Watson-Crick

A-UA-UG-CG-C

AAGAAG

3’3’ 5’5’

UUCUUC 3’3’5’5’

mRNAmRNA(Phe)(Phe)

tRNAtRNAPhePhe

Wobble positionWobble position

Variable positionVariable position

AAGAAG

3’3’ 5’5’

UUCUUC 3’3’5’5’

mRNAmRNA(Phe)(Phe)

tRNAtRNAPhePhe

Wobble positionWobble position

Variable positionVariable position

Deamination of G = IDeamination of G = IUUUUUU 3’3’5’5’

(Phe)(Phe)

Base pairing at the wobble positionBase pairing at the wobble position

• Inosinate (I) Inosinate (I) often found at 5’ often found at 5’ wobble positionwobble position

• I can form H I can form H bonds with A, C, bonds with A, C, or Uor U

• Anticodon with I Anticodon with I can recognize can recognize more than one more than one synonymous synonymous codoncodon

Some bacteria get by with only 31 tRNA (not 61)Some bacteria get by with only 31 tRNA (not 61)

Weaker H-bond: speeds up prot. Syn.Weaker H-bond: speeds up prot. Syn.

*

Protein synthesis :Protein synthesis :translationtranslation

Aminoacylation of tRNAAminoacylation of tRNA

Charging of tRNACharging of tRNA

InitiationInitiationChain elongationChain elongationTerminationTermination

By Aminoacyl-tRNA SynthetasesBy Aminoacyl-tRNA Synthetases

Structure of the Aminoacyl-tRNA Linkage

Linkage type ?

Aminoacylation of tRNA

The genetic code is translated by two adaptors: The first is the aminoacyl-tRNA synthetase. The linkage is through a high energy bond created with ATP.

*

The second adaptor is the tRNA itself whose anticodon forms base pairs with the appropriate mRNA codon. An error in either step causes wrong aa in peptide chain.

High energy bond

*

Protein Synthesis Proceeds by the Addition of an AA to the C-terminus of a polypeptide

Peptidyl Transferase

Energy stored in aminoacyl-tRNA used in formation of peptide Energy stored in aminoacyl-tRNA used in formation of peptide bondbond

5’ 3’mRNA

Aminoacyl-tRNA Synthetases• Aminoacyl-tRNA - amino acids are covalently attached to the Aminoacyl-tRNA - amino acids are covalently attached to the

3’ end of each tRNA molecule 3’ end of each tRNA molecule (named as: alanyl-tRNA(named as: alanyl-tRNAAlaAla))

• Most species have at least Most species have at least 20 different aminoacyl-tRNA20 different aminoacyl-tRNA synthetasessynthetases

(1 per amino acid) (1 per amino acid)

• Each synthetase specific for a particular amino acid, but may Each synthetase specific for a particular amino acid, but may recognize isoacceptor tRNAsrecognize isoacceptor tRNAs

• Aminoacyl-tRNAs are high-energy molecules (the amino acid Aminoacyl-tRNAs are high-energy molecules (the amino acid has been “activated”)has been “activated”)

• The activation of an amino acid by aminoacyl-tRNA The activation of an amino acid by aminoacyl-tRNA synthetase requires ATP synthetase requires ATP

Amino acid + tRNA + ATPAmino acid + tRNA + ATP

Aminoacyl-tRNA + AMP + PPAminoacyl-tRNA + AMP + PPii

Synthetase binds ATP and correct AASynthetase binds ATP and correct AABased on Based on sizesize//chargecharge//hydrophobicityhydrophobicity

Synthetase selectively binds tRNA based onSynthetase selectively binds tRNA based onStructural featuresStructural featuresAnticodonAnticodonAcceptor stemAcceptor stem

Ester linkageEster linkage

Specificity of Aminoacyl-tRNA SynthetaseSpecificity of Aminoacyl-tRNA Synthetase

• Attachment of the correct amino acid to the corresponding Attachment of the correct amino acid to the corresponding tRNA is a critical steptRNA is a critical step

• Synthetase binds ATP and the correct amino acid (based on Synthetase binds ATP and the correct amino acid (based on size, charge, hydrophobicity)size, charge, hydrophobicity)

• Synthetase then selectively binds specific tRNA molecule Synthetase then selectively binds specific tRNA molecule based on based on structural features, anticodon and acceptor structural features, anticodon and acceptor stemstem

• Some aa-tRNA synthetases can proofreadSome aa-tRNA synthetases can proofread

• Isoleucyl-tRNA synthetase may bind valine Isoleucyl-tRNA synthetase may bind valine instead of isoleucine and form valyl-adenylateinstead of isoleucine and form valyl-adenylate

• The valyl-adenylate is usually then hydrolyzed The valyl-adenylate is usually then hydrolyzed to valine and AMP so that valyl-tRNAto valine and AMP so that valyl-tRNAIleIle does does not form not form

Proofreading Activity of Aminoacyl-tRNA SynthetasesProofreading Activity of Aminoacyl-tRNA Synthetases

1 in 1001 in 100

1 in 100001 in 10000

RibosomesRibosomesProtein synthesisProtein synthesis by a complex by a complex composed ofcomposed of the the ribosomeribosome

Ribosome moves 5’ to 3’ along mRNARibosome moves 5’ to 3’ along mRNA

Peptide grows N to C directionPeptide grows N to C direction

Initiation complex assembles at Initiation complex assembles at first mRNA codon, and first mRNA codon, and disassembles at termination stepdisassembles at termination step

Catalyzes peptide bond formationCatalyzes peptide bond formation

Assist ribosomeAssist ribosome

Carries infoCarries info

Carries activated AACarries activated AA

charged charged tRNAtRNA molecules molecules

mRNAmRNA

accessory accessory proteinprotein factors factors

*

Ribosomes Are Composed of Both rRNA and Protein

Small subunitSmall subunit Large subunitLarge subunit

30S30S 50S50S

Nobel Prize in Chemistry 2009Nobel Prize in Chemistry 2009

RibosomesRibosomes

Ribosomes Contain Two Aminoacyl-tRNA Binding Ribosomes Contain Two Aminoacyl-tRNA Binding SitesSites

• Ribosome must Ribosome must align two align two charged tRNA charged tRNA molecules so molecules so that anticodons that anticodons interact with interact with correct codons correct codons of mRNAof mRNA

• Aminoacylated Aminoacylated ends of the ends of the tRNAs are tRNAs are positioned at positioned at the site of the site of peptide bond peptide bond formationformation

• Ribosome must Ribosome must hold both mRNA hold both mRNA and growing and growing polypeptide polypeptide chainchain

30S30S

50S50S

Mechanism of TranslationMechanism of Translation

1. Initiation

2. Elongation

3. Termination

• The translation complex is assembled at the The translation complex is assembled at the beginning of the mRNA coding sequencebeginning of the mRNA coding sequence

• Complex consists of: Complex consists of: Ribosomal subunitsRibosomal subunitsmRNA template to be translatedmRNA template to be translatedInitiator tRNA moleculeInitiator tRNA moleculeProtein initiation factorsProtein initiation factors IF-1IF-1

IF-2IF-2IF-3IF-3

Initiator tRNAInitiator tRNA

• First codon translated is usually AUGFirst codon translated is usually AUG

• Each cell contains at least two methionyl-Each cell contains at least two methionyl-tRNAtRNAMet Met molecules which recognize AUGmolecules which recognize AUG

The The initiator tRNAinitiator tRNA recognizes initiation recognizes initiation codonscodons

formylformylmethioninemethionine

Second Second tRNAtRNAMetMet recognizes only internal AUG recognizes only internal AUG

•BacteriaBacteria: : N-formylmethionyl-tRNAN-formylmethionyl-tRNAffMetMet

(Eukaryotes(Eukaryotes: : methionyl-tRNAmethionyl-tRNAiiMetMet))

EF-Tu interaction (does not bind formyl-tRNAEF-Tu interaction (does not bind formyl-tRNAffMetMet) )

IF2 interacts with formyl-tRNAIF2 interacts with formyl-tRNAffMetMet

Shine-Dalgarno sequences in Shine-Dalgarno sequences in E. coliE. coli mRNAmRNA

• Ribosome-binding sites at the 5’ end of mRNA for Ribosome-binding sites at the 5’ end of mRNA for several several E. coliE. coli proteins proteins

• In prokaryotes, the 30S ribosome binds to a region of In prokaryotes, the 30S ribosome binds to a region of thethe

mRNA (mRNA (Shine-Dalgarno sequenceShine-Dalgarno sequence) upstream of the ) upstream of the initiationinitiation

sequencesequence S-D sequence binds to a complementary base S-D sequence binds to a complementary base sequence sequence at the 3’ end of the 16S rRNAat the 3’ end of the 16S rRNA

• Complementary base pairing of S-D Complementary base pairing of S-D sequencesequence

Initiator tRNA moleculeInitiator tRNA molecule

Initiation of TranslationInitiation of Translation

Protein initiation factorsProtein initiation factors

IF-1IF-1IF-2IF-2IF-3IF-3

BacteriaBacteria:: N-formylmethionyl-tRNAN-formylmethionyl-tRNAffMetMet

Second Second tRNAtRNAMetMet recognizes only internal AUG recognizes only internal AUG

Inititation Inititation factors factors IF1, IF2, IF1, IF2, and IF3 and IF3 are are required to required to form the form the ribosomal ribosomal complexcomplex

Formation of the prokaryotic 70S initiation factorFormation of the prokaryotic 70S initiation factor *

Translation Initiation in EukaryotesTranslation Initiation in Eukaryotes

• Eukaryotic initiation factor 4 (eIF-4), (or cap binding Eukaryotic initiation factor 4 (eIF-4), (or cap binding protein, CBP) binds to the (5’ end) 7methylguanylate cap protein, CBP) binds to the (5’ end) 7methylguanylate cap of eukaryotic mRNAof eukaryotic mRNA

• A A preinitiationpreinitiation complexcomplex forms (40S ribosome, forms (40S ribosome, aminoacylated aminoacylated initiator tRNAinitiator tRNA, other factors) and , other factors) and searches the mRNA 5’ 3’ for an initiator codonsearches the mRNA 5’ 3’ for an initiator codon

• The The Met-tRNAMet-tRNAiiMetMet binds to AUG, and the 60S ribosomal binds to AUG, and the 60S ribosomal

subunit binds to complete the complexsubunit binds to complete the complex

• The initiator tRNA is in the P siteThe initiator tRNA is in the P site

• Site A is ready to receive an aminoacyl-Site A is ready to receive an aminoacyl-tRNAtRNA

• Elongation is a three-step cycle:Elongation is a three-step cycle:

(1) Positioning the correct aa-tRNA in site (1) Positioning the correct aa-tRNA in site A A (2) Formation of a peptide bond (2) Formation of a peptide bond (3) Shifting mRNA by one codon (3) Shifting mRNA by one codon

Chain Elongation is a Three-Step Chain Elongation is a Three-Step MicrocycleMicrocycle

Translating an mRNA Molecule--Elongation

Translocation

Positioning the correct aa-tRNA in site APositioning the correct aa-tRNA in site A

Formation of a peptide bondFormation of a peptide bond

Shifting mRNA by one codonShifting mRNA by one codon

three-step cycle:three-step cycle:

Insertion of aa-tRNA Insertion of aa-tRNA by EF-Tu during chain by EF-Tu during chain elongationelongation

PositioningPositioning of the aminoacyl-tRNA of the aminoacyl-tRNA *

Cycling of EF-Tu-GTPCycling of EF-Tu-GTP *

*

• Substrate Substrate bindingbinding site in site in 23S rRNA and 23S rRNA and 50S ribosomal 50S ribosomal proteinsproteins

• CatalyticCatalytic activityactivity from 23S rRNA from 23S rRNA (an RNA-(an RNA-catalyzed catalyzed reaction)reaction)

Peptidyl TransferasePeptidyl Transferase Catalyzes Peptide Bond Formation Catalyzes Peptide Bond Formation

•Peptidyl transferasePeptidyl transferase activity is contained within the large ribosomal subunit activity is contained within the large ribosomal subunit

CatalyticCatalytic activityactivity from 23S rRNA (an RNA-catalyzed from 23S rRNA (an RNA-catalyzed reaction)reaction)

Adenine: abstracts proton Adenine: abstracts proton donates proton donates proton

*

A Pocket in the 23S Ribosomal RNA is the Catalyst for the Peptidyl Transferase Activity

N3 of the Adenine in the catalytic pocket of 23S rRNA abstracts a proton from the aa acylated to the tRNA. The Amino N of the aa acid then attacks the carboxyl group of the peptide in the P site.

The protonated Adenine donates its hydrogen to the Oxygen linked to the tRNA thus releasing the tRNA.

tRNA originally attached to peptide in the P site is released

New Amino Acid

• Translocation Translocation stepstep: the new : the new peptidyl-tRNA is peptidyl-tRNA is moved from the A moved from the A site to the P site, site to the P site, while the mRNA while the mRNA shifts by one shifts by one codoncodon

• The The deaminoacylated deaminoacylated tRNA has shifted tRNA has shifted from the P site to from the P site to the E site (exit the E site (exit site)site)

TranslocationTranslocation Moves the Ribosome by One Moves the Ribosome by One CodonCodon

EF-G-GTP

GTP hydrolysis causeslarge conformationalchange that moves peptidyl tRNA to P site

*

the mRNA shifts by one codonthe mRNA shifts by one codon

Translational Elongation in Prokaryotes

EF-Tu is the most abundant protein in E. coli -- ~6% of total protein. It is a G protein.

P site

A site

A/P hybrid site

Hydroysis of GTP by EF-G causes large conformational change that moves tRNA from the A/P hybrid site to the P site.

EF-G is another G protein and hydrolysis of its GTP powers translocation.

E site(exit site)

Formation of the peptide chainFormation of the peptide chain

• Growing peptide chain Growing peptide chain extends from the peptidyl-extends from the peptidyl-tRNA (P site) through a tunnel tRNA (P site) through a tunnel in the 50S subunitin the 50S subunit

• Newly synthesized Newly synthesized polypeptide does not begin to polypeptide does not begin to fold until it emerges from the fold until it emerges from the tunneltunnel

• Elongation in eukaryotes is Elongation in eukaryotes is similar to E. coli:similar to E. coli: EF-1aEF-1a - docks the aa-tRNA - docks the aa-tRNA into A siteinto A site EF-1bEF-1b - recycles EF-1a - recycles EF-1a EF-2EF-2 - carries out - carries out translocationtranslocation

Termination of TranslationTermination of Translation

• E. coliE. coli release factors: RF-1, RF-2, RF-3 release factors: RF-1, RF-2, RF-3

• Translocation positions one of three Translocation positions one of three termination codons in A site: UGA, UAG, UAAtermination codons in A site: UGA, UAG, UAA

• No tRNA molecules recognize these codons No tRNA molecules recognize these codons and protein synthesis stallsand protein synthesis stalls

• One of the release factors binds and causes One of the release factors binds and causes hydrolysis of the peptidyl-tRNA to release the hydrolysis of the peptidyl-tRNA to release the polypeptide chainpolypeptide chain

• Four phosphoanhydride bondsFour phosphoanhydride bonds are cleaved for each are cleaved for each amino acid added to a polypeptide chainamino acid added to a polypeptide chain

AminoAmino acidacid activationactivation:: Two ~P bonds Two ~P bonds

ATP ATP AMP + 2 PAMP + 2 Pii

ChainChain elongationelongation:: Two ~P bonds Two ~P bonds

2 GTP 2 GTP 2 GDP + 2 P 2 GDP + 2 Pii

More energy then req for formation of single peptide bondMore energy then req for formation of single peptide bond

Compensate for loss of entropyCompensate for loss of entropy

Protein Synthesis is Energetically ExpensiveProtein Synthesis is Energetically Expensive

Specific order of aa in peptideSpecific order of aa in peptide

Specific aa linked to tRNASpecific aa linked to tRNA

Specific tRNA/codon interactionSpecific tRNA/codon interaction

Final exam

1-45 new material

46-75 old material

10 questions from each previous exam