2013 10 31-From DNA to Protein

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Chapter 6

From DNA to Protein:How Cell Read the Genome



Genetic information directs the synthesis of protein

The central dogma of molecular biology



Genes can be expressed with different efficienciesUntranscribed portions



Nucleotide



The chemical structure of RNA differs slightly

from that of DNA

3’

5’

phosphodiester

bond



Uracil forms a base pair with adenine

2 hydrogen bonds



RNA molecules can form intramolecular base pairs

and fold into specific structures

Base-pair with complementary sequences

Conventional

base-pair interactions

Nonconventional

base-pair

interactions



Non-Coding strand Anti-sense strand

Template strand

Transcription produces an RNA complementary

to one strand of DNA

Coding strand

Sense strand



Sense & antisense strand



DNA duplication by DNA polymerase



Transcription by RNA polymerase



DNA is transcribed by the enzyme RNA polymerase

ATP

CTP

UTPGTP



Transcription can be visualized in the electron microscope

Gene 1 Gene 2

rRNAs

RNA polymeraseDNA

Ribosomal proteins



RNA polymerase vs DNA polymerase

RNA polymerase DNA polymerase

Ribonucleotides Deoxyribonucleotides

Without primer With primer

1/104 error rate 1/107 error rate



Types of RNA produced in cells

messenger RNA

ribosomal RNA

microRNA

transfer RNA

n o n - m e s s e

n g e r R N A



Signals in the sequence of a gene tell bacteria

RNA polymerase where to start and stop transcription

Bacterial RNA polymerase

Chain elongation



Bacterial RNA polymerase

Bacterial promoters and terminators

have specific nucleotide sequences

that are recognized by RNA polymerase



The direction of transcription is determined by the orientation of the promoter at the beginning of each gene

Some genes are transcribed using

one strand DNA as a template, whereas others are

transcribed using the other DNA strand



The three RNA polymerases in eucaryotic cells

mRNA

sRNAs

sRNAs

T b i i i i RNA l II



To begin transcription, eucaryotic RNA polymerase II

requires a set of general transcriptional factors

-25

TATA-binding protein

Dramatic local distortion in the DNA

Dephosphorylated form

Transcription initiation complex

Phosphorylate the tail

of RNA polymerase II

Allow the template strandto be exposed by

ATP hydrolysis

TATA bi di t i (TBP) bi d t TATA b



TATA-binding protein (TBP) binds to TATA box sequences

and distorts the DNA

TATA box DNA

TBP (TATA-binding protein)

B f th b t l t d RNA l l d



Before they can be translated, mRNA molecules made

in the nucleus move out into the cytoplasm via pore

in the nuclear envelope

Pores in

nuclear envelope

TEM

Ph h l ti f RNA l II ll



(1) Capping

(2) Polyadenylation

(3) Splicing

Phosphorylation of RNA polymerase II allows

RNA-processing proteins to assemble on its tail

TFIIH

Eucaryotic mRNA molecules are modified by




capping and polyadenylation (1)

Start after 25 nucleotides

has been polymeized

(1) To increase the stability of the eucaryotic mRNA molecule

(2) To aid its export from the nucleus to the cytoplasm

(3) To identify the RNA molecule as an mRNAFunctions:





capping and polyadenylation (2)

1

2 34

5

CH3

Eucaryotic and bacterial genes are organized differently



Eucaryotic and bacterial genes are organized differently

Promoter

Intron is longer than exon

Most human genes are broken into exons and introns



Most human genes are broken into exons and introns

3 exons

26 exons

Special nucleotide sequences signal



Special nucleotide sequences signal

the beginning and the end of an intron

RNA splicing might occurred before or after polyadenylation

R: A or G

Y: C or U

N: A or C or G or U

long short

Branch point of the lariat

intron-exon

boundary (border)

RNA splicing



RNA splicing

An introns forms a branched structure during splicing



An introns forms a branched structure during splicing

5’

3’ 5’

Branch point of the lariat

Spliceosome



Spliceosome

Small nuclear RNAs (snRNAs) + Proteins

= Small nuclear ribonucleoprotein particles

(snRNPs)

The -tropomyosin gene can be spliced in different ways



The tropomyosin gene can be spliced in different ways

(1) Many different protein to be produced from the same gene by alternative splicing(2) 60% of human genes probably undergo alternative splicing

A specialized set of RNA-binding proteins signal that



A specialized set of RNA binding proteins signal that

a mature mRNA is ready for export to the cytoplasm

Recognizes and exports

only completed mRNAs

Life times depends on

(1) Nucleotide sequence (3’ untranslated sequence)

(2) The type of cell

Exon junction complex (EJC)

Mark completed RNA splices

3’ untranslated region



3 untranslated region

Procaryote and eucaryotes handle



y y

their RNA transcripts differently

Transcription in procaryotic or eucaryotic cells (1)



p p y y ( )

Transcription in procaryotic or eucaryotic cells (2)



p p y y ( )

Bacteria Eucaryotic cells

Single type Three types (I, II, III)

X General transcription factors

Short Long

RNA polymerase

Accessory proteins

Nontranscribed DNA between genes

Small RNAs



(1) siRNA (small interfering RNA)

(2) miRNA (microRNA)(3) piRNA (piwi-interacting RNA)

miRNA



siRNA



The nucleotide sequence of an mRNA is translated into



the amino acid sequence of a protein

via the genetic code

Stop

codonsStart

codon

T

h r e e - n u c l e o t i d e

c o d o n s

A m i n o

a c i d s

UUU codes for phenylalanine



Heinrich MatthaeiMarshall Nirenberg

Messages of mixed repeating sequences further



narrowed the coding possibilities

Gobind Khorana

The Nobel Prize in Physiology or Medicine 1968



Interpretation of the genetic code and its function in protein synthesis

An RNA molecule can be translated

i th ibl di f



in three possible reading frames

Reading frame



Frame shift



tRNA molecules are molecular adaptors,

linking amino acids to codons



linking amino acids to codons

pseudouridine

dihydrouridine

L-shape molecule

Wobble base-pairing



61 codons

31 anticodons 20 amino acids

The genetic code is translated by means of two adaptors

that act one after another



that act one after another

Aminoacyl-tRNA synthase

Charged tRNA

Charging

12 Anticodon

Ribosomes are found in the cytoplasm of a eucaryotic cell



Attached to the ER

TEM

Free in thecytosol

Ribosome

A ribosome is a large complex of four RNAs and

more than 80 proteins



more than 80 proteins

(rRNA)

(rRNA)

Catalyzes the formation

of polypeptide chain

Matches the tRNA to

the codon of the mRNA

1/3

2/3

Each ribosome has a binding site for mRNA

and three binding sites for tRNA



and three binding sites for tRNA

Large subunit Small subunit

Large subunit

Small subunit

Exit

peptidyl-tRNA

aminoacyl-tRNA

Translation takes place in a four-step cycle



Catalyzed by an enzymatic site

in the large subunit

Large subunit

Ribosomal RNAs give the ribosome its overall shape



Protein

Large subunit of a bacterial ribosome

Catalytic site for peptide bond formation

Rate of sedimentation in an ultracentrifuge

Initiation of protein synthesis in eucaryotes requires

initiation factors and a special initiator tRNA



initiation factors and a special initiator tRNA

Start codon

E P A

E P A

E P A

or formylmethionine in bacteria

only this charged RNA

can binds to the P-site

In the final phase of protein synthesis, the binding of

release factor to an A-site bearing a stop codon



release factor to an A site bearing a stop codon

terminates translation

Stop codons

UAG

UGA

UAA

A single procaryotic mRNA molecule can encode

several different proteins



several different proteins

Operons



Proteins are translated by polyribosomes



Polyribosomes (Polysomes)

Inhibitors of procaryotic protein synthesis

are used as antibiotics



The proteosome degrades short-lived and

misfolded proteins



Active site of

the proteases

Ubiquination for protein degradation



Ubiquitin

Protein production in a eucaryotic cell

requires many steps



Many proteins require additional modification

to become fully functional



Glycosylation (> 100 kinds)

An RNA world may have existed

before modern cells arose



An RNA molecule can in principle guide

the formation of an exact copy of itself



Ribozyme

Ribozyme – RNA molecules that possess catalytic activity



Ribozyme – RNA molecules that possess catalytic activity

A ribozyme is an RNA molecule that possesses

catalytic activity



Biochemical reactions that can be catalyzed by ribozymes



Could an RNA molecule catalyze its own synthesis?



RNA may have preceded DNA and proteins in evolution

2013 10 31-From DNA to Protein

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