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Molecular Genetics-223Spring 2012

nature of the genetic code

maintenance of genes through DNA replication

transcription of information from DNA to mRNA

translation of mRNA into protein

DNA repair

Recombination

Intron splicing

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Brenner S., Jacob F., and Meselson M. (1961)

An unstable intermediate carrying information fromgenes to ribosomes for protein synthesis.

Nature 190: 576-581 (13 May 1961)

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RNA

Composition

StructureFunction

Stability

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Chemistry of Information

N

NNH

N

NH2

HN

NNH

N

O

H2N

N

NH

NH2

O

NH

NH

O

O

adenine (A) guanine (G) cytosine (C) uracil (U)

Nucleoside bases found in RNA:

Nucleic acids are polymers ofnucleotides.Each nucleotide includes a nucleoside base

that is either

a purine (adenine or guanine), or

a pyrimidine (cytosine, uracil, or thymine).

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RNA species may contain modified bases. Examples:

N

NNH

N

NH2

HN

NNH

N

O

H2N

N

NH

NH2

O

NH

NH

O

O

adenine (A) guanine (G) cytosine (C) uracil (U)

N

NNH

N

NH2

H3C

+HN

NNH

N

O

H2N

CH3

+

N

NH

NH2

O

CH3+NH

NH

HN

O

O

1-methyladenine (m1A) 7-methylguanine (m7G) 3-methylcytosine (m3C) pseudouracil ()

Nucleoside bases found in RNA:

Examples of modified bases found in tRNA:

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In a nucleotide, e.g., adenosine monophosphate (AMP),

the base is bound to a ribose sugar, which has a phosphate

in ester linkage to the 5' hydroxyl.

N

NN

N

NH2

adenine adenosine adenosine monophosphate (AMP)

O

OHOH

HH

H

CH2

H

HO

N

NNH

N

NH2

N

NN

N

NH2

O

OHOH

HH

H

CH2

H

OO3P2

ribose

5'

adenine

4'

3' 2'

1'

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Nucleic acids

have a backbone

of alternating Pi &ribose moieties.

Phosphodiester

linkages form as

the 5' phosphateof one nucleotide

forms an ester link

with the 3' OH of

the adjacent

nucleotide.

A short stretch of

RNA is shown.

N

NN

N

NH2

O

OHO

HH

H

CH2

H

ribose

adenine

P

O

O OO

OHO

HH

H

CH2

H

N

N

NH2

O

P

O

O O

OP

O

O

O

cytosine5'

4'

3' 2'

1'

ribose3'

5'

3'end

5'end

(etc)

nucleic acid

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Hydrogen bonds link 2 complementary nucleotide bases

on separate nucleic acid strands, or on complementary

portions of the same strand.Conventional base pairs: A & U (or T); C & G.

In the diagram at left, H-bonds are in red. Bond lengths

are inexact. The image is based on X-ray crystallography

of tRNAGln. H atoms are not shown.

N

NNH

N

O

N

N

NH

N

O

H

H

H

H

H

guanine(G)

cytosine(C)

G

C

G C base

pair in tRNA

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Secondary structure

Base pairing over extended stretches of complementary

base sequences in two nucleic acid strands stabilizes

secondary structure, such as the double helix of DNA.

Stacking interactions between adjacent hydrophobic

bases contribute to stabilization of such secondary

structures. Each base interacts with its neighbors aboveand below, in the ladder-like arrangement of base pairs

in the double helix, e.g., of DNA.

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Double helical stems arise from base pairing between

complementary stretches of bases within the same strand.

Loops occur where lack of complementarity, or thepresence ofmodified bases, prevents base pairing.

RNA structure:Most RNAs have secondary structure, consisting of

stem & loop domains.

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The cloverleaf model of

tRNA secondary structureemphasizes the 2 major types

of secondary structure,

stem and loop domains.

tRNAs typically include many modified bases,

particularly in the loop domains.

Tertiary structure depends on interactions of bases at

more distant sites. Many of these interactions involvenon-standard base pairing and/or interactions involving

three or more bases.

tRNAs usually fold into an L-shaped tertiary structure.

anticodon loop

acceptorstemtRNA

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The amino acid

attaches to the ribose

of the terminal A

(in red) at the 3' end.The anticodon loop is

at the opposite end of

the L shape.

anticodon

acceptor

stem

tRNAPhe

anticodon loop

acceptorstemtRNA

Extending out from the

"acceptor stem", the 3' end of

every tRNA has the sequence CCA.

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Tertiary base pairs

#46

(m7G)

#22G #13

C

Tertiary base

pairs in tRNAPhe

#46

(m7G)

#22

G

#13

C

Tertiary base

pairs in tRNAPhe

Non-standard H bond interactions, some linking 3 bases,

help stabilize the L-shaped tertiary structure of tRNA. H

atoms are not shown.

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Some other RNAs,

including viral RNAs &

segments of ribosomalRNAs, fold in

pseudoknots, tertiary

structures that mimic the

3D structure of tRNA.

Pseudoknots are

stabilized by tertiary

(non-standard) H-bondinteractions.

anticodon

acceptorstem

tRNAPhe

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Aminoacyl-tRNA Synthetases catalyze linkage of theappropriate amino acid to each tRNA. The reaction occurs

in two steps.

In step 1, an O atom of the amino acid a-carboxyl attacks

the P atom of the initial phosphate of ATP.

O

OHOH

HH

H

CH2

H

OPOPOP

O

O

O

O

O O

O

R

H

C C

NH3+

O

O

O

OHOH

HH

H

CH2

H

OPOC

O

O

H

CR

NH2

O

Adenine

Adenine

ATPAmino acid

Aminoacyl-AMP

PPi

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In step 2, the

2' or 3' OH of

the terminal

adenosine of

tRNA attacks

the amino acidcarbonyl C

atom.

O

OHOH

HH

H

CH2

H

OPOC

O

O

H

CR

NH2

O

Adenine

O

OHO

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

C

HC

O

NH3+

R

tRNA

AMP

Aminoacyl-AMP

Aminoacyl-tRNA

(terminal 3nucleotide

of appropriate tRNA)3 2

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Aminoacyl-tRNA Synthetase

Summary of the 2-step reaction:

1. amino acid + ATP aminoacyl-AMP + PPi

2. aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP

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There is a different Aminoacyl-tRNA Synthetase

(aaRS) for each amino acid.

Each aaRS recognizes its particular amino acid and thetRNAs coding for that amino acid.

Domains of tRNA recognized by an

aaRS are called identity elements.

Most identity elements are in theacceptor stem & anticodon loop.

Aminoacyl-tRNA Synthetases arose

early in evolution

anticodon loop

acceptorstemtRNA

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One of the current models of translation start

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Key enzymes: 1) Guanylyl transferase,2) Guanine-7-methyltransferase,

3) 2-O-metyltransferase.

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An example to viral mechanisms

blocking eukaryotic translation

Prevention of cap-dependent translation.

Viral protease (2A-pro) cleaves eIF4G.

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Firefly luciferase mRNA (with 5 and 3 UTRs from a plant gene)

was electroporated into protoplasts. At intervals, the amount of

luciferase mRNA was checked (to determine half-life), and the

amount of luciferase activity (which reflects the amount of

translation product).

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Pre-mRNA Polyadenylation

Most cytoplasmic mRNAs have a polyAtail (3 end) of 50-250 Adenylates

Added post-transcriptionally by anenzyme, Poly(A) Polymerase (PAP)

Turns over (recycles) in cytoplasm

y

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Functions of the PolyA

Tail

1. Promotes mRNA stability- Deadenylation (shortening of the polyA

tail) can trigger rapid degradation of

the mRNA

2. Enhances translation- promotes recruitment by ribosomes

- bound by a polyA-binding protein inthe cytoplasm called PAB1

- synergistic stimulation with Cap!

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Overview of PolyadenylationMechanism

1. Transcription extendsbeyond mRNA end

2. Transcript is cut at 3 end ofwhat will become themRNA (in green)

3. PolyA Polymerase adds~250 As to 3 end

4. Extra RNA (in red)

degraded

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Eukaryotic cytoplasmic ribosomes are larger and more

complex than prokaryotic ribosomes.

Ribosome

Source

Whole

Ribosome

Small

Subunit

Large

Subunit

E. coli 70S 30S

16S RNA

21 proteins

50S

23S & 5S

RNAs

31 proteinsRat

cytoplasm

80S 40S

18S RNA

33 proteins

60S

28S, 5.8S, &5S

RNAs

49 proteins

Ribosome Composition (S = sedimentation coefficient)

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Structure of theE. coli Ribosome

The cutaway view at right shows positions of tRNA (P, E

sites) & mRNA (as orange beads, data from Joachim

Frank lab at the Wadsworth Center)

small subunit

large subunit

mRNA

location

EF-G

tRNA

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The cutaway view at right shows that the tunnel in the

yeast large ribosome subunit, through which nascentpolypeptides emerge from the ribosome, lines up with the

lumen of the ER Sec61 channel.

small

subunit large

subunit

Sec61 channel

path of

nascentprotein