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

phosphodiester

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

UTPGTP

Transcription can be visualized in the electron microscope

Gene 1 Gene 2

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

T b i i i i RNA l II

To begin transcription, eucaryotic RNA polymerase II

requires a set of general transcriptional factors

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

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

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)

Eucaryotic and bacterial genes are organized differently

Promoter

Intron is longer than exon

Most human genes are broken into exons and introns

3 exons

26 exons

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

An introns forms a branched structure during splicing

3’ 5’

Branch point of the lariat

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

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)

The nucleotide sequence of an mRNA is translated into

the amino acid sequence of a protein

via the genetic code

codonsStart

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

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

Aminoacyl-tRNA synthase

Charged tRNA

Charging

12 Anticodon

Ribosomes are found in the cytoplasm of a eucaryotic cell

Attached to the ER

Free in thecytosol

Ribosome

A ribosome is a large complex of four RNAs and

more than 80 proteins

(rRNA)

Catalyzes the formation

of polypeptide chain

Matches the tRNA to

the codon of the mRNA

Each ribosome has a binding site for mRNA

and three binding sites for tRNA

Large subunit Small subunit

Large subunit

Small subunit

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

Start codon

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

A single procaryotic mRNA molecule can encode

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

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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