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Information decoding: Interpreting the genetic code

Genes VIII. Chapter 7Molecular biology; Chapter

Genetic code

DNA sequence of single gene is colinear with a.a sequence of polypeptide (Fig.9.1; Weaver p567)

F. Crick and S. Brenner (1961) deletion or insertion mutation of bacteriophage T4 DNA , found that:

- a group of three bases codes for one amino acid

- the code is not overlap

- the base seq is read from a fixed starting point without punctuation

- the code is degenerate

- nucleotide sequence (5＇->3＇) is exactly the same order of a.a. sequence of coded polypeptide (N terminal to C

terminal)

Q. mRNA carry genetic message, but how is it read?

Genetic code is nonoverlopping, nonpuntuation

- overlapping code- punctuated code- unpunctuates code (triplet code)

(Weaver Fig. 18.3, lane 5)

Genetic codeSpecific proteins can be made in cell-free systems-

M.W. Nirenberg (1961)

- tRNA, fMet-tRNAFMet

- ATP & GTP

- aminoacyl synthetase

- free ribosome subunits & polyribosome

contain mRNA

- amino acid

- Mg+2

- PolyU (UUU) coded for polyphenylalanine

- polypeptide is made & release from ribosome

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Genetic codeSpecific proteins can be made in cell-free systems-M.W. Nirenberg (1961)

- tRNA, fMet-tRNAFMet

- ATP & GTP- aminoacyl synthetase- free ribosome subunits & polyribosome contain

mRNA- amino acid- Mg+2- PolyU (UUU) coded for polyphenylalanine- polypeptide is made & release from ribosome

Codons: the sequence of mRNA is read in groups of three nucleotides (triplet), each codon specify a particular a.a. (Fig. 9.1)

- but UAG, UGA, UAA do not encode an amino acid, termination codon (stop codons)

- AUG codes for methionin (Met) - first AUG is read by ribosome in a mRNA called

initiationcodon (start codon)

Codon- all eukaryotic polypeptide start with methionine- all prokaryotic polypeptide start with modified

methionine (N-formylmethionine) RNA is composed of 4 types of nucleotides: A,U,G, C. each codon contain 3 nt (triplet)

43 = 64 possible (triplet) codons- only 20 amino acid are commonly found in protein- each amino acid is coded by several different

codons

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Genetic code is “degenerated”

- only methionine(AUG) and tryptophan(UGG) are represented by a single codon

- because of “degeneracy”, point mutation change a single nt in DNA

- hence only change single nt in mRNA, often NOeffect on a.a sequence

- to minimize the deleterious effects of mutations

Codon family- first two bases (same) sharing with different 3rd

bases (A,U,G, C). e.g . UA family; UAU, UAC(Tyr), UAA (stop),

UAG (Stop)unmixed family: e.g. CC family,

CCU,CCA,CCG,CCC (=Pro)mixed family : UU family

UUU,UUC (=Phe)UUA,UUG (=Leu)

Fig. 9.33rd-base relationship same meaning No. Of Codons3rd base irrelavent U,C,A,G 32 codons(8 families)

Purine A,G 12 codons (6 pairs*UAA,UAG= stop)

Pyrimidine U,C 14 codons (7 pairs)

Three out of four U,C,A 3 codon (AUX=Ile)

Unique definition G only 2 (AUG=Met)(UUG=Trp)A only 1 (UGA=stop)

Synonyms

codons specify the same a.a.- most synonyms differ only in the third base of codon

- GUU, GUC, GUA, GUG all code for valine- each codon in mRNA is recognized (base

pairing; complementary) by a triplet bases (anticodon) in tRNA

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anticodon -3’X’-Y’-Z’5’- (tRNA) codon -5’X –Y-Z 3’ - (mRNA)

---- (reversed direction)

codon 5’-XYZ-3’ / anticondon 5’-Z’Y’X’-3’

Ambiguity: UUU (Phe), at high [Mg+2] (UUU Leu)

Ala tRNA discover

Robert W. Holley

1968 Nobel laureate

tRNAis unique among nucleic acid, tRNA contain“unusual” bases modification is after incorperatedinto polyribonucleotide chain

Pyrimidines(U,C)Uridine- ribothymidine(T), dihydrouridine(D),

pseudouridine (y) 4- thiouridine(S4U)Cytidine- 3-methylcytidine, 5-methylcytidine

Purines(A,G)-Adenosine- inosine, N6-mehtyladenosine(m6A),

N6-isopentenyladenosine(I6A)Guanosine- 7-methylguanosine, Queuosine(Q),

Wyosine(Y)

Extra loop (arm): (variable loop);(44th~48th)

- class 1 tRNA 3-5 nt- class 2 tRNA 13-21 bases, ~5 bases pairing

* additional bases 47:1 via 47:18

D (DHU) arm: (14th-21st nt) the 4th loop: 8-12 unpaired bases, contain a modified base “dihydro-U” (also name D loop)- extra nt (17:1(17,18); 20:1 & 20:2 (20, 21)-aminoacyl-tRNA recognize tRNAat this region

tRNA contains modified bases that influence its pairing properties

(Fig. 7.6)

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General rules of codon-anticodon pairing

- first two bases of codon are AU, 3rd base prefer C rather than U (more stable codon-anitcodonpair preferred)

- U and A usually are not employed at 1st base of anticodon

- U converted to modified form (at 5’ wobble site) of anticodon prefer for A over G in 3rd base of codon

- I (at 5’ wobble site) of anticodon prefer pair with U or C than with A

The Wobble Hypothesis

The first two bases of the codon make normal (canonical) H-bond pairs with the 2nd and 3rd bases of the anticodonAt the remaining position, less stringent rules apply and non-canonical pairing may occur The rules: first base U can recognize A or G, first base G can recognize U or C, and first base I can recognize U, C or A (I comes from deamination of A) Advantage of wobble: dissociation of tRNA from mRNA is faster and protein synthesis too

Wobble hypothesis

- 5’ end of anticodon is not as spatial confined as the other two

- allow it to form H-bond with several bases at 3’end of a codon

wobble base pairing- base paring of the third base of codon is less stringent than first two bases

wobble position: the third position of codon (at mRNA)

- some single tRNA can base pair with more than one codon

- phe tRNA had anticodon GAA, recognize UUU, UUC codon

- mismatch in pairing in third codon is often tolerated

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The Purpose of Wobble

Although wobble also contribute codonrecognition & specificityWobble position’s H-bond weakerKinetic advantage to wobbleCodon-anticodon less stable, t-RNA dissociate from mRNA easlierAccelerate the process protein synthesis

Codon-anticodon pairing involved wobble

Table 9.1, Fig 9.4,51st base in anticodon(5’ end) base in codon(3rd bases)

G U or CC GA UU (thiouracil) A or GI (inosine) A,U, or C

*I= Inosine (A deamination)

How many codon can be recognized depends on 1st base of anticodon (5’)?

1st anticodons base:A or C can recognize one codonG or U can recognize 2 codonI can recognize 3 codons

University of the genetic code

“universal code” - all organisms use same genetic code

- a few differences, in mitochondria & chloroplasts

- same codons has different meaning

Natural variation in the standard genetic codon

- mitochondria(Table )- prokaryotics (Mycoplasma UGA=Trp; E coliUGA Selenocysteine; Ciliated protozoa UAA, UGA=Glu)

- some lower eukaryotes

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tRNA are charged with amino acids by individuals synthetase

An aminoacyl-tRNA synthetase (aaRS) charges tRNA with an amino acid

AA-tRNA synthetase

Aminoacyl-tRNA synthetase (aaRS) reaction (aminoacylation)

- specific enzymes recognize specific a.a.- Cells have > 20 different aminoacyl-tRNAsynthetase, one for each amino acid

- Attachment to tRNA by aminoacyl-tRNAsynthetase activates the amino acid

Two-step activation process

1. AA-AMP bound to aminoacyl-tRNA synthetase- release pyrophospate (~pp) (Fig. 9.7)- ~P energy for reaction

2. AA~AMP transfer a.a. attach to 2’OH of ribose of adenosine- a.a. link by an ester bond from its carboxyl group

to a hydroxyl group of ribose of A(CCA end of a specific tRNA) become to aminoacyl-tRNA

- release AMP*esterfication is ATP-dependent

tRNA aminoacylation

aminoacyl-tRNA synthetaseAA + ATP <-------------------------> AA~AMP + P-P

aminoacyl-tRNA synthetaseAA~AMP + tRNA <-----------------> AA~tRNA + AMP

Charged tRNA : a tRNA carrying its cognate a.a.AA~tRNA formation is very accurate

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tRNA synthetase has 2 classes:

Class I synthetase:N -termus C terminus

Catalytic domain : ATP binding site & a.a. binding sites- 2 a.a. seq. (signature seq)- nucleotide binding motif fold(anti-parallel b-sheet)- signature seq containing ATP binding site

t-RNA acceptor helix binding sitetRNA anticodon binding domainOligomer formation domain

Two classes of aminoacyl-tRNAsynthetase (aaRS)

There are two classes of aminoacyl-tRNA synthetase(aaRS), which differ both in tertiary and quaternary structure

The 20 amino acids are equally divided between the two classes.

Class II synthetase:tRNA anticodon binding domain- (at N terminus)t-RNA acceptor helix binding site- surrounded by catalytic domain

Oligomer formation domain- various

Different of Class I & 2 aaRS

a.a for class I has larger & hydrophobic

a.a. specificity

Charge 3’OHAminoacylate tRNA’s 3’terminal 2’OH

Site of animoacylation

Several don’t interact with their bound tRNA’s anticodon

aaRS recognize its cognate tRNA

Anticodonrecognition

Lack class I seqHas a 7-strand β sheet

HVGH & KMSKS seq.(signature seq.)

Structuralmotifs

Class I Class II

The N-terminal sequence which folds into the Rossmann nucleotide binding pocket contains the conserved class I signature sequence motifs as HVGH andKMSKS and the C-terminal sequence contains proline and tryptophan residues

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aaRS tRNA synthetase-tRNA binding

enzyme-tRNA binding along the L-shape of tRNA

Classes I synthetase contact tRNAapproach from D loopcontact at minor groove of acceptor stem & at anticodonloopU1-A72 base pair is disruptedacceptor stem lies in deep pocket in proteinATP binds near acceptor stemanticodon loop is distorted at U35-U36

Class II synthetase:Class II synthetases( e.g. Aspartyl-tRNA synthetase(AspRS) Class II aaRS have active sites built on a antiparallel βsheet surrounded by α-helices. The protein interacts with the major groove of the tRNA acceptor arm

- tRNA anticodon binding domain- (at N terminus)

- t-RNA acceptor helix binding site- surrounded by catalytic domain

- Oligomer formation domain- various

Class II synthetase contact tRNAat major groove of acceptor helix& at anticodon loopATP bind to terminal A residuesingle-stranded tail lies deep in proteinanticodon loop is distorted

Accuracy of tRNA synthetaserecognize correct

t-RNA & amino acidadmittance - scrunity, rejection/

acceptance cycle- Proofreading !

Class II synthetase contact tRNA

at major goove of acceptor helix & at anticodonloopATP bind to terminal A residuesingle-stranded tail lies deep in proteinanticodon loop is distorted

Accuracy of tRNA synthetase recognize correct t-RNA & amino acidadmittance - scrunity, rejection/acceptance cycle- proofreading

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tRNA synthetase-tRNA binding

enzyme-tRNA binding along the L-shape of tRNA

Classes I synthetase contact tRNAapproach from D loopcontact at minor groove of acceptor stem & at anticodon loopU1-A72 base pair is disruptedacceptor stem lies in deep pocket in proteinATP binds near acceptor stemanticodon loop is distorted at U35-U36

2 important recognition steps for accurate translation

1. Correct a.a. must be selected for covalent attachment a tRNA by an aminoacyl-tRNA synthetase (aaRS)

2. Correct aminoacyl-rRNA must pair with an mRNA codon at the ribosome

Second genetic code

How does an aaRS recognize a tRNA so that it can be charged with proper amino acid?aaRS recognize unique structural features of tRNA

- aminoacyl-tRNA synthetase recognize tRNA’sacceptor stem, D loop & anticodon stem

- Contact enzyme-tRNA face

Recognition of tRNAs

by the aminoacyl-tRNA synthetasesAnticodon region is not the only recognition site The "inside of the L" and other regions of the tRNA molecule are also important Read pages 1080-1082 on specificity of several aminoacyl-tRNA synthetases

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Accuracy synthesize charged tRNA(kinetic proofreading)

1. Recognize correct tRNA by synthetase (Fig. 9.12)- synthetase greater affinity for its cognate (correct)

tRNA rapidly, dissociates slowly- cogate tRNA trigger change in conformation- aminoacylation occurs rapidly

if incorrect tRNA attend - synthetase associate with incorrect tRNA slowly;

dissociation quickly- aminoacylation slow

2. Accuracy synthesize aminoacly-tRNA(chemicalproofreading) (Fig. 9. 13)

synthetase bind a.aadenylation (AMP) tRNA binding to synthetase

if synthetase bind to wrong amino acid proofreading occurproofreading 1: aminoacyl-adenylate is hydrolyzed

if charging with tRNA at synthetase formproofreading 2: aminoacyl-tRNA is hydrolyzed

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Double sieve mechanismproofreading & editing by amino-tRNA

synthetases

Avoid producing tRNA with wrong a.a. attached

Synthetase have 2 sites:First sieve: activation site - reject larger a.a.Second sieve: Editing (hydrolytic) side – fine sieve, reject which smaller a.a.Wrong a.a-AMP hydrolyzed to AMP + a.a.Only correct one converted to aa-tRNA

Figure 19.38 Double sieve of isoleucine-tRNA synthetase

IleRS: first sieve : exclude too large a.a. (if too large(phe) or wrong shape (leu)Smaller a.a. such as valine also can through IleRS first sieve, when transport to editing site (second sieve), proofreading or editing will degrade it

Open reading frame (ORF)

- a mRNA read in goups of 3 nt from 5’ end, it can read 3 possible “ reading frame” , depends onwhich nt is used as first base of first codon

- usually, only one reading frame will produce a functional protein

- the other two reading frame will include several stop codons

- correct reading frame is set in vivo by recognition by the ribosome of the initiation codon (AUG)

- AUG & GUG as initiation codons- AUG codes for methionine

RF example

RF1 U U A U G A G C G C U A A A ULeu Stop Ala Leu Asn

RF2 U U A U G A G C G C U A A A UTyr Glu Arg Stop

RF3 U U A U G A G C G C U A A A UMet Ser Ala Lys

- usually one sequence encodes only a single protein- but some bacteriophage several genes overlap, each gene has several RF

Suppressor tRNA mutated anticodon

Supressor genes: genes that cause suppressionof mutations in other genes

- Supressor mutation can reside in same or a different gene

- suppressor genes upsets the reading of the genetic code, misreading of specific nonsense or missense codon

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suppressor mutation create an aminoacyl-tRNAwith mutant anticodon can recognize terminal codon (insert an a.a.)

Nonesense mutation: any change of three triplet to UAG, UAA, UGA that cause termination of protein synthesis, cause premature chaintermination

Nonsense suppressor

a gene coding for a mutant tRNA can respond to one or more nonsense codons (Fig 9.14)nonsense suppression involves mutant tRNA (alter anticodon of tRNA)- most mutation involve single-point mutationamber suppressor tRNA recognize UAGochre suppressor tRNA recognize UAA

tRNA wild type suppressorcodon/anti codon/anti

trp UGG/CCA UGG/UCAUGA

Missense: change a codon for a specific a.a. to a codon specific for another a.a. (Fig. 9.15)- tRNA anticodon mutate but still carry same a.a.

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tRNA may influence the reading frame

Frameshift suppression (Figure 9.17)- mutant tRNA by insert extra base 3 bases to 4

bases- frameshit glycine tRNA anticodon(CCC

CCCC)- tRNA read 4 bases at a time- bacterial tRNALys suppressor recognize AAAA

or AAAU

Accuracy of translation (proofreading)

wrong GTP-AA-tRNA-EF-Tu complex get into A site, abnormal codon recognition causes GTP to be hydrolized to GDP & Pi, allow EF-Tudissociate from complex- weak H-bond of inncorect tRNA to a mRNA cause tRNA leave A site very quickly

- one a.a. misincorporated into polypeptide chain for every104 a.a polymerized

Ribsomal mutations also affect reading accuracy

- 30S ribosomal protein influence accuracy reading

- a missense mutation in a gene coding a ribosomal protein acts as supressor mutation (distort ribosome)

- incorrect a.a-tRNA is used for elogatepolypeptide chain

Antibiotics streptomycin

bind to 30S and disturb normal mRN-tRNA-ribosome interaction - S12 of ribosome involve misreading gentic code- slippery a.a.-tRNA move +1 or -1 base to pair with anticodon

- ribosome delay allow a.a.-tRNA rearrange its pairing