Section O

•Molecular Biology Course

Section PSection PTHE GENETIC CODETHE GENETIC CODEAND tRNA: AND tRNA: introduction to introduction to translationtranslation

•P1 THE GENETIC CODEP1 THE GENETIC CODE

•P2 tRNA STRUCTURE P2 tRNA STRUCTURE AND FUNCTIONAND FUNCTION

THE GENETIC CODETHE GENETIC CODE

NatureNatureDecipheringDecipheringFeatureFeatureEffect of muEffect of mu

tationtation

UniversalityUniversalityORFsORFsOverlapping Overlapping

genesgenes

NaturNaturee1.1. Genetic codeGenetic code is a triplet codeis a triplet code

(three nucleotide encode one amino (three nucleotide encode one amino acid)acid)

The way in which the nucleotide The way in which the nucleotide sequence in nucleic acids specifies sequence in nucleic acids specifies the amino acid sequence in proteins. the amino acid sequence in proteins.

The triplet codons are The triplet codons are nonoverlappingnonoverlapping and and comma-lesscomma-less. .

------UCU UCC CGU GGU GAAUCU UCC CGU GGU GAA------

2. Genetic code2. Genetic code is degenerate :is degenerate :

Only 20 amino acids are encoded by 4 Only 20 amino acids are encoded by 4 nucleotides in triplet codons nucleotides in triplet codons (4(433 =64 =64 of amino acids could potentially be of amino acids could potentially be encoded)encoded). Therefore, more than one . Therefore, more than one triplet are used to specify a amino triplet are used to specify a amino acids, and the genetic code is said to acids, and the genetic code is said to be be degeneratedegenerate, or to have , or to have redundancyredundancy..

DecipherinDecipheringg

System A: cell-free protein System A: cell-free protein synthesizing systemsynthesizing system from from E. coliE. coli

1.1.cell lysate treated by DNase to cell lysate treated by DNase to prevent new transcription prevent new transcription

2.2.Add homopolymeric synthetic Add homopolymeric synthetic mRNAs [poly(A)] mRNAs [poly(A)] ++ 19 cold (non- 19 cold (non-labeled) and one labeled aminoacidslabeled) and one labeled aminoacids

3.3.In vitro translationIn vitro translation4.4.Analyze the translated polypeptides Analyze the translated polypeptides

poly(U) ---UUU--- poly(U) ---UUU--- polyphenylalaninepolyphenylalanine

poly(C) ---CCC--- polyprolinepoly(C) ---CCC--- polyproline

poly(A) ---AAA--- polylysinepoly(A) ---AAA--- polylysine

poly(G) --- did not work because poly(G) --- did not work because of the complex secondary of the complex secondary structurestructureRandom co-polymers (e.g. U and G Random co-polymers (e.g. U and G

in the same RNA) were used as in the same RNA) were used as mRNAs in the cell-free system to mRNAs in the cell-free system to determine the codon for many determine the codon for many amino acids.amino acids.

System 2: Synthetic System 2: Synthetic trinucleotidestrinucleotides (late 1960s) could (late 1960s) could assign specific triplets assign specific triplets unambiguously to specific amino unambiguously to specific amino acids.acids.

Synthetic trinucleotides attach to the Synthetic trinucleotides attach to the ribosome and bind their corresponding ribosome and bind their corresponding aminoacyl-tRNAsaminoacyl-tRNAs from a mixture. Upon from a mixture. Upon membrane filtration, the trinucleotides membrane filtration, the trinucleotides bound with ribosome and aminoacyl-bound with ribosome and aminoacyl-tRNA would be retained.tRNA would be retained.

DecipherinDecipheringg

Fig(1)

FeatureFeaturessSynonymous codonsSynonymous codons:: 18 out of 20 amino acids have more than 18 out of 20 amino acids have more than

one codon to specify them, causing the one codon to specify them, causing the redundancy of the genetic code.redundancy of the genetic code.

the third positionthe third position::

pyrimidine ----synonymous (all cases)pyrimidine ----synonymous (all cases)

purine ----synonymous (most cases)purine ----synonymous (most cases)

the second positionthe second position::

pyrimidine ----hydrophilic amino acidspyrimidine ----hydrophilic amino acids

purine -----polar amino acids purine -----polar amino acids

Effect of Effect of MutationMutation

1. Transition1. Transition: : the most common the most common mutation in naturemutation in nature

changes from changes from purine to purine, or purine to purine, or pymidine to pymidinepymidine to pymidine

At third positionAt third position: no effect except for : no effect except for Met Met Ile; Trp Ile; Trp stop stopsecond positionsecond position: results in similar : results in similar

chemical type of amino acids.chemical type of amino acids.

2. Transversions:2. Transversions: purine purine pymidine pymidine At At third position: third position: over half over half

have no effect and result in a have no effect and result in a similar type of amino acid. similar type of amino acid. (Example: (Example: Asp Asp Glu Glu))

At At second position: second position: change change the type of amino acid.the type of amino acid.

In the first positionIn the first position, mutation , mutation (both transition and transvertion) (both transition and transvertion) specify a specify a similar typesimilar type of amino of amino acid, and in a few cases it is the acid, and in a few cases it is the same amino acidsame amino acid..

Thus, natural triplet codons are Thus, natural triplet codons are arranged in a way to minimize arranged in a way to minimize the harmful effect of an the harmful effect of an mutation to an organism. mutation to an organism.

UniversalitUniversalityy•The standard codons are true for The standard codons are true for

most organisms, but not for all. most organisms, but not for all. CodonCodon Usual Usual

meaningmeaningAlternativeAlternative Organelle or Organelle or

organismorganism

AGA AGGAGA AGG ArgArg Stop,SerStop,Ser Some animal Some animal mitochondriamitochondria

AUAAUA IleIle MetMet MitochondriaMitochondria

CGGCGG ArgArg TrpTrp Plant mitochondriaPlant mitochondria

CUNCUN LeuLeu ThrThr Yeast Yeast mitochondriamitochondria

AUU GUG AUU GUG UUGUUG

Ile Val Leu Ile Val Leu StartStart Some protozoansSome protozoans

UAA UAGUAA UAG StopStop GluGlu Some protozoansSome protozoans

UGAUGA StopStop TrpTrp Mitochondria,mycoplasMitochondria,mycoplasmama

ORFsORFs

Open reading frames (ORFs) Open reading frames (ORFs) are are suspected coding regions suspected coding regions starting starting withwith ATG ATG and end with and end with TGA,TAATGA,TAA or or TAGTAG identified by computer. identified by computer.

When the ORF is known to encode When the ORF is known to encode a certain protein, it is usually a certain protein, it is usually referred as a referred as a coding regioncoding region..

Overlapping Overlapping genesgenes•Generally these occur where the Generally these occur where the

genome size is small (viruses in genome size is small (viruses in most cases) and there is a need for most cases) and there is a need for greater information storage density.greater information storage density.

•More than one start codons in a More than one start codons in a DNA sequence are used for DNA sequence are used for translate different proteins. translate different proteins.

•A way to maximize the coding A way to maximize the coding capability of a given DNA sequence.capability of a given DNA sequence.

tRNA STRUCTURE AND FUNCTION

tRNA primary structure

tRNA secondary structure

tRNA tertiary structure

tRNA function

Aminoacylation of tRNAs

Aminoacy-tRNA synethetases

Proofreading

tRNAs charging

•tRNAtRNA are the are the adaptoradaptor molecules molecules that deliver amino acids to the that deliver amino acids to the ribosome and decode the ribosome and decode the information in information in mRNAmRNA..

• Linear lengthLinear length: : 60-9560-95 nt (commonly nt (commonly 7676))

• Residues: Residues: 1515 invariant invariant and and 88 semi-semi-invariantinvariant .The position of invariant and .The position of invariant and semi-variant nucleosides play a role in semi-variant nucleosides play a role in either the either the secondary and tertiarysecondary and tertiary structure. structure.

tRNA primary tRNA primary structurestructure

• Modified bases:Modified bases:

Sometimes accounting for Sometimes accounting for 20%20% of of the total bases in one tRNA the total bases in one tRNA molecule.Over molecule.Over 5050 different types of different types of them have been observed. them have been observed. Fig(1)Fig(1)

tRNA secondary tRNA secondary structurestructure

•The The cloverleafcloverleaf structure is a structure is a common secondary structural common secondary structural representation of tRNA molecules representation of tRNA molecules which shows the base paring of which shows the base paring of various regions to form various regions to form four four stems (arms)stems (arms) and and three loopsthree loops. .

Fig(2)Fig(2)

Figure 2, tRNA secondary structure

D loopD loop T loopT loop

Anticodon loopAnticodon loop

• The 5’-and 3’-The 5’-and 3’-end are largely end are largely base-paired to base-paired to form the amino form the amino acid acceptor acid acceptor stem which stem which has no loophas no loop. .

•Amino acid acceptor stem:Amino acid acceptor stem:

Composed of Composed of 3 3 oror 4 4 bp stem bp stem and a loop and a loop called the called the D-D-loop (DHU-loop (DHU-loop)loop) usually usually containing the containing the modified base modified base dihydrouracil.dihydrouracil.

•D-arm and D-loopD-arm and D-loop

Consisting of a Consisting of a 55 bp stem and a bp stem and a 77 residues loop in residues loop in which there are which there are three adjacent three adjacent nucleosides called nucleosides called the the anticodonanticodon which are which are complementary to complementary to the codon the codon sequence sequence (a triplet (a triplet in the mRNA)in the mRNA) that that the tRNA the tRNA recognize. recognize.

Anticodon loop:Anticodon loop:

•Variable arm and T-arm:Variable arm and T-arm:

Variable arm:Variable arm: 3 to 21 3 to 21 residues and may form residues and may form a stem of up to 7 bp.a stem of up to 7 bp.

T-armT-arm is composed of a is composed of a 5 bp stem ending in a 5 bp stem ending in a loop containing the loop containing the invariant residues invariant residues GTGTC. C.

tRNA tertiary tRNA tertiary structurestructure

•Formation:Formation:

9 hydrogen bones 9 hydrogen bones (tertiary (tertiary hydrogen bones) hydrogen bones) to help the to help the formation of tRNA tertiary formation of tRNA tertiary structure, mainly involving in the structure, mainly involving in the base paring between the base paring between the invariant bases. invariant bases.

•Hydrogen bonds:Hydrogen bonds:

• Base pairing between residuesBase pairing between residues in in the D-and T-arms fold the tRNA the D-and T-arms fold the tRNA molecule over into an molecule over into an L-shapeL-shape, with , with the anticodon at one end and the the anticodon at one end and the amino acid acceptor site at the amino acid acceptor site at the other. The base pairing is other. The base pairing is strengthened by strengthened by base stacking base stacking interactions. interactions.

Fig (3)Fig (3)

tRNA functiontRNA function

•When charged by attachment of When charged by attachment of a specific amino acid to their a specific amino acid to their 3’-3’-endend to become to become aminoacyl-tRNAsaminoacyl-tRNAs, , tRNA molecules act as adaptor tRNA molecules act as adaptor molecules in protein synthesis. molecules in protein synthesis.

Aminoacylation of Aminoacylation of tRNAstRNAs• Reaction step:Reaction step:First, the aminoacyl-First, the aminoacyl-

tRNA synthetase tRNA synthetase attaches AMP to attaches AMP to the-COOH group of the-COOH group of the amino acid the amino acid utilizing ATP to utilizing ATP to create create anaminoacyl anaminoacyl adenylate adenylate intermediate.intermediate.

Then, the Then, the appropriate tRNA appropriate tRNA displaces the AMP. displaces the AMP.

• Nomenclature of tRNA-Nomenclature of tRNA-synthetases and charged tRNAssynthetases and charged tRNAs

catalyze amino acid-tRNA joining reaction catalyze amino acid-tRNA joining reaction which is extremely specific.which is extremely specific.

Amino acid: serineAmino acid: serine

Cognate tRNA: tRNACognate tRNA: tRNAserser

Cognate aminoacyl-tRNA synthetase: Cognate aminoacyl-tRNA synthetase:

seryl-tRNA synthetaseseryl-tRNA synthetase

Aminoacyl-tRNA: seryl-tRNAAminoacyl-tRNA: seryl-tRNAserser

Aminoacyl-tRNA synthetasesAminoacyl-tRNA synthetases

•The synthetase enzymes are either The synthetase enzymes are either monomers, dimersmonomers, dimers or one of two or one of two types of types of tetramertetramer.They contact .They contact their cognate tRNA by the inside of their cognate tRNA by the inside of its L-shape and use certain parts of its L-shape and use certain parts of the tRNA, called the tRNA, called identity elements, identity elements, to distinguish these similar to distinguish these similar molecules from one another. molecules from one another. Fig(4)Fig(4)

ProofreadinProofreadingg

• ProofreadingProofreading occurs at occurs at step 2step 2 when a when a synthetase carries out synthetase carries out step 1step 1 of the of the aminoacylation reaction with the aminoacylation reaction with the wrong, but chemically similar, amino wrong, but chemically similar, amino acid. acid.

• Synthetase will not attach the Synthetase will not attach the aminoacyl adenylate to the cognate aminoacyl adenylate to the cognate tRNA, but hydrolyze the aminoacyl tRNA, but hydrolyze the aminoacyl adenylate instead.adenylate instead.

ThanksThanks

Fig （ 1 ） Modified nucleosides in tRNA

fig(3) tRNA tertiary structure

Fig(4) Identity elements in various tRNA molecules

Identity element: They are particular parts

of the tRNA molecules.These are not always the anticodon sequence,but base pair in the acceptor stem.If these are swapped between tRNAs then the synthetases enzymes can be tricked into adding the amino acid to the wrong tRNA

P1 The genetic code

UniversalityModifications of the genetic code

CodonCodon Usual Usual meaningmeaning

AlternativeAlternative Organelle or Organelle or organismorganism

AGA AGGAGA AGG A rgA rg Stop,SerStop,Ser Some animal Some animal mitochondriamitochondria

AUAAUA IleIle MetMet MitochondriaMitochondria

CGGCGG ArgArg TrpTrp Plant,mitochondriaPlant,mitochondria

CUNCUN LeuLeu ThrThr Yeast mitochondriaYeast mitochondria

AUU GUG AUU GUG UUGUUG

Ile Val Leu Ile Val Leu StartStart Some protozoansSome protozoans

UAA UAGUAA UAG StopStop GluGlu Some protozoansSome protozoans

UGAUGA StopStop TrpTrp Mitochondria,mycoplasMitochondria,mycoplasmama

The universal genetic code

First First position(position(5’end)5’end)

Second positionSecond position Third Third positiposition(3’eon(3’end)nd)

UU CC AA GG

UUPhePhe

PhePhe

LeuLeu

LeuLeu

UUUUUU

UUCUUC

UUAUUA

UUGUUG

SerSer

SerSer

SerSer

SerSer

UCUUCU

UCCUCC

UCAUCA

UCGUCG

TyrTyr

TyrTyr

StopStop

stopstop

UAUUAU

UACUAC

UAAUAA

UAGUAG

CysCys

CysCys

StopStop

TrpTrp

UGUUGU

UGCUGC

UGAUGA

UGGUGG

UU

CC

AA

GG

CCLeuLeu

LeuLeu

LeuLeu

LeuLeu

CUUCUU

CUCCUC

CUACUA

CUGCUG

ProPro

ProPro

ProPro

ProPro

CCUCCU

CCCCCC

CCACCA

CCGCCG

HisHis

HisHis

GlnGln

GlnGln

CAUCAU

CACCAC

CAACAA

CAGCAG

ArgArg

ArgArg

ArgArg

ArgArg

CGUCGU

CGCCGC

CGACGA

CGGCGG

UU

CC

AA

GG

AAIleIle

IleIle

IleIle

MetMet

AUUAUU

AUCAUC

AUAAUA

AUGAUG

ThrThr

ThrThr

ThrThr

ThrThr

ACUACU

ACCACC

ACAACA

ACGACG

AsnAsn

AsnAsn

LysLys

LysLys

AAUAAU

AACAAC

AAAAAA

AAGAAG

SerSer

SerSer

ArgArg

ArgArg

AGUAGU

AGCAGC

AGAAGA

AGGAGG

UU

CC

AA

GG

GGValVal

ValVal

ValVal

ValVal

GUUGUU

GUCGUC

GUAGUA

GUGGUG

AlaAla

AlaAla

AlaAla

AlaAla

GCUGCU

GCCGCC

GCAGCA

GCGGCG

AspAsp

AspAsp

GluGlu

GluGlu

GAUGAU

GACGAC

GAAGAA

GAGGAG

GlyGly

GlyGly

GlyGly

GkyGky

GGUGGU

GGCGGC

GGAGGA

GGGGGG

UU

CC

AA

GG

Figure 6 tRNA tertiary structure

Figure 7 tRNA tertiary structure