Chapter 7

Genetic code

• 7.1 THE GENETIC CODE

• 7.2 tRNA STRUCTURE AND FUNCTION

THE GENETIC CODE

NatureDeciphering(破

译 )

FeatureEffect of mutation

UniversalityORFsOverlapping

genes

Nature

1. Genetic code is a triplet code

(three nucleotide encode one amino acid)

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

The triplet codons are nonoverlapping（不重叠） and comma-less（无逗号） .

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

2. Genetic code is degenerate （简并性） :

Only 20 amino acids are encoded by 4 nucleotides in triplet codons (43 =64 of amino acids could potentially be encoded). Therefore, more than one triplet are used to specify a amino acids, and the genetic code is said to be degenerate, or to have redundancy（有丰余） .

Deciphering

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

1. cell lysate treated by DNase to prevent new transcription

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

3. In vitro translation4. Analyze the translated polypeptides

poly(U) ---UUU--- polyphenylalanine

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

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

poly(G) --- did not work because of the complex secondary structure

Random co-polymers (e.g. U and G in Random co-polymers (e.g. U and G in the same RNA) were used as mRNAs the same RNA) were used as mRNAs in the cell-free system to determine the in the cell-free system to determine the codon for many amino acids.codon for many amino acids.

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

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

Deciphering

密码子的破译• 遗传密码的破译是六十年代分子生物学最辉煌的成就。

• 50 年代的数学推理过程和 61-65 的实验研究阶段。

• 1954 年 物理学家 George Gamov 根据 DNA 中的 4 种

核苷酸，在蛋白中存在 20 种氨基酸的对应关

系。 41=4; 42=16 少于 20 。

• 那么 43=64 ； 44=256

随后的实验证明了他的假设是正确的。

① 在体外无细胞蛋白合成体系中加入人工合成的 polyU (1961, Nirenberg, Matthaei)

密码子的破译

② 混合共聚物实验 - 对密码子中碱基组成的测定—polyAC 8 种不同的密码子（ 1963 ， Speyer, Ochoa)

③AA-tRNA 与确定的密码子结合（ 1964 ， Nirenberg,和 Leder)

④ 用重复共聚物破译密码（ Nishimura,Jones 和Khorana ）用有机化学合成法合成特定的重复序列。

Fig(1)

Features

Synonymous codons: 18 out of 20 amino acids have more than one codon

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

the third position:

pyrimidine ----synonymous (all cases)

purine ----synonymous (most cases)

the second position:

pyrimidine ----hydrophilic amino acids

purine -----polar amino acids

Effect of Mutation

1. Transition （转换） : the most common mutation in nature

changes from purine to purine, or pymidine to pymidine

At third position: no effect except for Met Ile; Trp stopsecond position: results in similar chemical type

of amino acids.

2. Transversions （颠换） : purine pymidine At third position: over half have no

effect and result in a similar type of amino acid. (Example: Asp Glu)

At second position: change the type of amino acid.

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

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

Universality （通用性）

• The standard codons are true for most organisms, but not for all.

CodonCodon Usual Usual meaningmeaning

AlternativeAlternative 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

ORFs （可读框） Open reading frames (ORFs) are

suspected coding regions starting with ATG and end with TGA,TAA or TAG identified by computer.

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

Overlapping genes

• Generally these occur where the genome size is small (viruses in most cases) and there is a need for greater information storage density.

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

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

• 重叠基因：是指一个基因的编码区部分或全部与另一个基因的编码区重叠。

• ATG GTC GGG GAC CGA TGT TTG GAA

• ATG TTT GGA

tRNA STRUCTURE AND FUNCTION

tRNA primary structure

tRNA secondary structure

tRNA tertiary structure

tRNA function

Aminoacylation of tRNAs

Aminoacy-tRNA synethetases

Proofreading

tRNAs charging

• tRNA are the adaptor molecules that deliver amino acids to the ribosome and decode the information in mRNA.

• Linear length: 60-95 nt (commonly 76)

• Residues: 15 invariant and 8 semi-invariant .The position of invariant and semi-variant nucleosides play a role in either the secondary and tertiary structure.

tRNA primary structure

• 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.

tRNA secondary structure

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

tRNA secondary structure

D loopD loop T loopT loop

Anticodon loopAnticodon loop

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

•Amino acid acceptor stem:Amino acid acceptor stem:

Composed of 3 or 4 bp stem and a loop called the D-loop (DHU-loop) usually containing the modified base dihydrouracil（二氢尿嘧啶） .

•D-arm and D-loopD-arm and D-loop

Consisting of a 5 bp stem and a 7 residues loop in which there are three adjacent nucleosides called the anticodon which are complementary to the codon sequence (a triplet in the mRNA) that the tRNA recognize.

Anticodon loop:Anticodon loop:

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

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

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

tRNA tertiary structure

• Formation:

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

• Hydrogen bonds:

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

tRNA function

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

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

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

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

• Nomenclature of tRNA-synthetases and charged tRNAs

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

Amino acid: serineAmino acid: serineCognate tRNA: tRNACognate tRNA: tRNAserser

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

seryl-tRNA synthetaseseryl-tRNA synthetaseAminoacyl-tRNA: seryl-tRNAAminoacyl-tRNA: seryl-tRNAserser

Aminoacyl-tRNA synthetasesAminoacyl-tRNA synthetases

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

Proofreading

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

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

• 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

Anti-codon 及其两侧碱基修饰对密码子 解读的生物学意义

Xo5U (5- 羟基尿苷 )

Cmnm5U (5- 羧甲基氨甲基尿苷 )

mCm5U (5- 甲氧基羰甲基尿苷 )

Xm5s2U (5- 甲基 -2 硫代尿苷 )

K2C (2- 赖氨酸胞苷 )

Com5U (5(2)- 羟羧甲基尿苷 )

I (Inosine 次黄嘌呤 )

m7G (7- 甲基尿苷 )

m5C (5- 甲基胞苷 )

m6A (6- 甲基腺苷 )

s2C (2- 硫代胞苷 )

ψ ( 假尿苷 )

Um (2’-O- 甲基尿苷 )

Q (Queuosine)

a) Methylated Nt at anti-codon and flanked

b) 被修饰的 Nt34 的配对能力

Nt1 of anti-codon Nt3of codon

U (mt, ct) A,U,C,G

CmO5U (5(2)- 羟羧甲基尿苷 ) A,G,U

Cmnm5U (5- 羧甲基氨甲基尿苷 ) A,G

mCm5U (5- 甲氧基羰甲基尿苷 ) A,G

Um (2’-O- 甲基尿苷 ) A,G

Xm5S2U (5- 甲基 -2 硫代尿苷 ) A

Q (Queuosine) U,C

I (Inosine ） U,C,A

U* （ 4 硫代尿苷） G,A