3. DNA, RNA and Protein Synthesis

7/28/2019 3. DNA, RNA and Protein Synthesis

1/73

LOGO

3. DNA, RNA and Protein

Synthesis


2/73

Where it all began

You started as a cell smaller thana period at the end of a sentence

.


3/73

How did you

get from there

to here?

And now look at you


4/73

Cell cycle & cell division

Cell cycle life of a cell from

origin to division into

2 new daughter cells

Cell division

continuity of life =

reproduction of cells

reproduction unicellular organisms

growth

repair & renew


5/73

Dividing cell

Dividing cell duplicates DNA separates each copy to

opposite ends of cell

splits into 2 daughter cells

human cell duplicates ~3 meters DNA

separates 2 copies so each daughter cell has

complete identical copy

error rate = ~1 per 100 million bases


6/73

DNA Replication

The double helix unwinds into 2separate strands. The two

strands serve as templates for

copying two new strands of

DNA each new helix

contains 1 original strand plus 1new strand semi-

conservative replication with

normal base pairing: A with T,

and G with C.

The energy for this comes from

the nucleotide precursors.

They all have 3 phosphates on

them, like ATP, and 2 of the

phosphates are removed tomake the DNA.


7/73

Nucleotide precursors

ATPAdenosine triphosphate

++

modified nucleotide

adenine ribose + Pi + Pi + Pi


8/73

Bacterial chromosome replication

The separation ofcomplementary strands

occurs in the regions of

the DNA called origins

of replication (ori).

Bacterial chromosome

have a single origin.

The separated strandsform replication fork


9/73

Eukaryotic chromosome replication

Because of their large size, eukaryoticchromosome have multiple origins

Replication begins at specific sites

where the two parental strands

separate and form replication

bubbles.

The bubbles expand laterally, as

DNA replication proceeds in both

directions.

Eventually, the replication

bubbles fuse, and synthesis of

the daughter strands is

complete.

1

2

3

Origin of replication

Bubble

Parental (template) strand

Daughter (new) strand

Replication fork

Two daughter DNA molecules

In eukaryotes, DNA replication begins at many sites along the giant

DNA molecule of each chromosome.In this micrograph, three replication

bubbles are visible along the DNA of

a cultured Chinese hamster cell (TEM).

(b)(a)

0.25 m


10/73

DNA Replication

DNA gyrase

Addition of RNA primer (10-15 bp),

are synthesized by primase


11/73

Antiparallel Elongation

DNA polymerases cannot initiate the synthesis of apolynucleotide

They can only add nucleotides to the 3 end (5 3)

The initial nucleotide strand

is a primer = RNA sequences, are synthesized byprimase

3

5 3

RNA primer

newly synthesized DNA

5

5


12/73

RNA primer

5

3

3

5

3

5

direction of leading strand synthesis

direction of lagging strand synthesis

replication fork

Synthesis of the strands


13/73

5

3 5

3

Movement of the replication fork



14/73

Movement of the replication fork

RNA primer

Okazaki fragment

RNA primer

5



15/73

3

RNA primer

5

DNA polymerase III initiates at the primer andelongates DNA up to the next RNA primer

5

53

5

newly synthesized DNA (100-1000 bases)

(Okazaki fragment)

53

DNA polymerase I inititates at the end of the Okazaki fragment

and further elongates the DNA chain while simultaneously

removing the RNA primer with its 5 to 3 exonuclease activity

pol III

pol I


16/73

newly synthesized DNA

(Okazaki fragment)5

3

53

DNA ligase seals the gap by catalyzing the formation

of a 3, 5-phosphodiester bond in an ATP-dependent reaction


St d ti t th li ti f k iti


17/73

5

3

3

5

3

5

Strand separation at the replication fork causes positive

supercoiling of the downstream double helix

DNA gyrase is a topoisomerase II, which

breaks and reseals the DNA to introduce negative

supercoils ahead of the fork

Fluoroquinolone antibiotics target DNA gyrases in many

gram-negative bacteria: ciprofloxacin and levofloxacin (Levaquin)


18/73

Figure 16.16

Overall direction of replicationLeading

strand

Lagging

strand

Lagging

strand

Leading

strandOVERVIEW

Leading

strand

Replication fork

DNA pol III

Primase

PrimerDNA pol III Lagging

strand

DNA pol I

Parental DNA

5

3

43

2

Origin of replication

DNA ligase

1

5

3

Helicase unwinds theparental double helix.1

Molecules of single-

strand binding protein

stabilize the unwound

template strands.

2 The leading strand is

synthesized continuously in the

5 3 direction by DNA pol III.

3

Primase begins synthesis

of RNA primer for fifth

Okazaki fragment.

4

DNA pol III is completing synthesis of

the fourth fragment, when it reaches the

RNA primer on the third fragment, it will

dissociate, move to the replication fork,

and add DNA nucleotides to the 3 end

of the fifth fragment primer.

5 DNA pol I removes the primer from the 5 end

of the second fragment, replacing it with DNA

nucleotides that it adds one by one to the 3 end

of the third fragment. The replacement of the

last RNA nucleotide with DNA leaves the sugar-

phosphate backbone with a free 3 end.

6 DNA ligase bonds

the 3 end of the

second fragment to

the 5 end of the first

fragment.

7

A summary of DNA replication


19/73

Proteins That Assist DNA Replication

Table 16.1


20/73

Proteins That Assist DNA Replication :

DNA Polymerase

Prokaryote FunctionPol I Gap filling & DNA repair

Pol II Gap filling & DNA repair

Pol III Replication

Eukaryote

Pol Lagging strand replication

Pol Gap filling & DNA repair

Pol Leading strand replication

Pol Mitochondria replication

Pol Gap filling & DNA repair


21/73

Eukaryotic chromosomal DNA molecules Have at their ends nucleotide sequences, called

telomeres, that postpone the erosion of genes nearthe ends of DNA molecules

If the chromosomes of germ cells becameshorter in every cell cycle Essential genes would eventually be missing from the

gametes they produce

Figure 16.19 1 m

Replicating the Ends of DNA Molecules


22/73

Replicating the Ends of DNA Molecules

The ends ofeukaryotic

chromosomal DNA Get shorter

with each

round of

replication

An enzyme calledtelomerase

Catalyzes thelengthening of

telomeres in germcells

Figure 16.18

End of parentalDNA strands Leading strandLagging strand

Last fragment Previous fragment

RNA primer

Lagging strand

Removal of primers and

replacement with DNA

where a 3 end is available

Primer removed but

cannot be replaced

with DNA because

no 3 end available

for DNA polymerase

Second round

of replication

New leading strand

New lagging strand 5

Further rounds

of replication

Shorter and shorter

daughter molecules

5

3

5

3

5

3

5

3

3


23/73

From nucleus to cytoplasm

Where are the genes? genes are on chromosomes in nucleus

Where are proteins synthesized?

proteins made in cytoplasm by ribosomes How does the information get from nucleus to

cytoplasm?

messenger RNA

nucleus

C l D


24/73

Central Dogma

RNA

DNA

Protein

Transcription

Translation

Replication

Reverse

Transcription

Juang RH (2004) BCbasics


25/73

RNA

ribose sugar N-bases

uracil instead of thymine,

which U : A modified bases

single stranded

RNADNAtranscription


26/73

RNA polynucleotide chain

2 -OH makes3, 5 phosphodiester

bond unstable

DNA polynucleotide chain


27/73

Examples of modified bases found in RNA

Dihydrouridine Pseudouridine 1-methylguanosine 7-methylguanosine

1-methyladenosine 2-thiocytidine 5-methylcytidine Ribothymine


28/73

Types of RNA

In prokaryote cells :

- messenger RNA (mRNA) :

an exact copy of a gene

- ribosomal RNA (rRNA) :

short (1,500-4,700

nucleotides), components of

ribosome- transfer RNA (tRNA) :

molecule that transport

amino acids to the ribosome

during synthesis protein

In eukaryote cells


29/73

Transcription

Transcription Is the synthesis of RNA under the direction of DNA

Occurs only in a chromosome segments thatcontain genes, many copies

Transcribed DNA strand = template strand=antisense strand untranscribed DNA strand = coding strand= sense

strand

Synthesis of complementary RNA strand transcription bubble

RNA polymerase : unwinds the DNA helix and thencopies one strand of DNA to RNA

Template ( ) Strand Transcribes to mRNA


30/73

Template (-) Strand Transcribes to mRNA

3 5

5 3 RNA

template strand

nontemplate strand (+) strand

(-) strand

5 3

coding strand

AUG

Codon is denoted on mRNA

ATG

TAC

UAG

TAG

ATC

Startcodon

Identicalsequence



31/73

Transcription

Transcription bubble


32/73

MEKANISME TRANSKRIPSI

= aktivator, o=operator, p=promotor,tsp=situs inisiasi transkripsi, RBS=ribosom

binding site, ATG=kodon start, STOP=kodonstop, t=terminator

Komponen transkripsi : promotor, situs

inisiasi transkripsi, dan terminator


33/73

MEKANISME TRANSKRIPSI

Promotor : tempat RNA pol mengikatkan diri utkmemulai transkripsi (recognition site) menentukan kecepatan transkripsi

Terdiri dari 2 blok urutan nukleotida yg terpisah :

- blok 1 : ~6 pb, - 5 sampai - 8 (Pribnow box/TATA

box) dari situs inisiasi transkripsi- blok 2 : ~10 pb, - 35 dari situs inisiasi transkripsi

Situs inisiasi transkripsi : tempat awal transkripsi(+1).

Pengenalan & pengikatan RNA pol di blok 2membuat blok 1 membuka mjd untai tunggal.

Salah satu untai tunggal menjadi templat dantranskripsi dimulai di +1.


34/73


35/73

Transcription in Prokaryotes

Promoter sequences

RNA polymerase

molecules bound to

bacterial DNA


36/73


Initiation RNA polymerase binds to promoter sequence

on DNA


37/73


Elongation RNA polymerase unwinds DNA

~20 base pairs at a time

reads DNA 3 5

builds RNA 5 3 (the energy governs the synthesis!)

No proofreading

1 error/105 bases many copies

short life


38/73


Termination RNA polymerase stops at termination

sequence

mRNA leaves nucleus through pores

RNA GC

hairpin turn


39/73

Structure of prokaryotic mRNA

5

3

PuPuPuPuPuPuPuPu AUGShine-Dalgarno sequence initiation

The Shine-Dalgarno (SD) sequence base-pairs with a pyrimidine-rich

sequence in 16S rRNA to facilitate the initiation of protein synthesis

AAUtermination

translated region

T i i i E k


40/73

LOGO

Transcription in Eukaryotes


41/73

Prokaryote vs Eukaryote genes

Prokaryotes DNA in cytoplasm

circular chromosome

naked DNA

no introns

Eukaryotes DNA in nucleus

linear chromosomes

DNA wound on histone

proteins introns vs. exons

eukaryotic

DNA

exon = coding (expressed) sequence

intron = noncoding (in between) sequence

Intron and Exon in Eukaryotic Cells


42/73

Intron and Exon in Eukaryotic Cells

mRNA

DNA

5 3

cap

poly Atail

exon exonexonintron intron

mature mRNA

Processing

Transcription

Splicing

promotor3 5

Take place in nucleus

start codon stop codon

To cytoplasm

Intron deleted



43/73


Promoters signal theinitiation of RNA synthesis

Transcription factors Help eukaryotic RNA

polymerase recognize promoter

sequences Initiation complex

transcription factors bind topromoter region upstream ofgene

proteins which bind to DNA &turn on or off transcription

TATA box binding site

only then does RNApolymerase bind to DNA


44/73

Transcription initiation

Control regions on DNA promoter

nearby control sequence on DNA

binding of RNA polymerase & transcription factors

base rate of transcription

enhancers distant control

sequences on DNA

binding of activatorproteins

enhanced rate (high level)of transcription


45/73


3 RNA polymerase enzymes RNA polymerase I

only transcribes rRNA genes

RNA polymerase II transcribes genes into mRNA

RNA polymerase III

only transcribes rRNA genes

each has a specific promoter sequence it

recognizes

P i i l i


46/73

Post-transcriptional processing

Eukaryotic cells modify RNA after transcription

Enzymes in the eukaryotic nucleus Modify pre-mRNA in specific ways before the genetic

messages are dispatched to the cytoplasm

Modify pre-mRNA (primary transcript) :

- Protect mRNAfrom RNase enzymes in cytoplasm by alteration of mRNA ends

add 5' cap

add polyA tail

A modified guanine nucleotideadded to the 5 end

50 to 250 adenine nucleotidesadded to the 3 end

Protein-coding segment Polyadenylation signal

Poly-A tail3 UTRStop codonStart codon

5 Cap 5 UTR

AAUAAA AAAAAA

TRANSCRIPTION

RNA PROCESSING

DNA

Pre-mRNA

mRNA

TRANSLATIONRibosome

Polypeptide

G P P P

5 3


47/73

Add 5 Cap

5 cap plays a role in ribosomerecognition of the 5 end of the RNA

molecule during translation

P l d l ti


48/73

Polyadenylation

Polyadenylation : a string of adenine nucleotides around100- 300 nucleotides in length is added to the 3 end of

the mRNA

This tail protects mRNA from degradation in cytoplasm,

increasing its stability and availability for translation

A modified guanine nucleotideadded to the 5 end

50 to 250 adenine nucleotidesadded to the 3 end

Protein-coding segment Polyadenylation signal

Poly-A tail3 UTRStop codonStart codon

5 Cap 5 UTR

AAUAAA AAAAAA

TRANSCRIPTION

RNA PROCESSING

DNA

Pre-mRNA

mRNA

TRANSLATIONRibosome

Polypeptide

G P P P

5 3

P t t i ti l i


49/73

Post-transcriptional processing

RNA splicing Removes introns and joins exons

Figure 17.10

TRANSCRIPTION

RNA PROCESSING

DNA

Pre-mRNA

mRNA

TRANSLATION

Ribosome

Polypeptide

5 Cap

Exon Intron

1

5

30 31

Exon Intron

104 105 146

Exon 3Poly-A tail

Poly-A tail

Introns cut out and

exons spliced togetherCoding

segment

5 Cap

1 1463 UTR3 UTR

Pre-mRNA

mRNA

RNA li i


50/73

RNA splicing

Is carried out byspliceosomes in some cases

antibodies to snRNPs are

seen in the autoimmune

disease systemic lupus

erythematosus (SLE)

Ribozymes

Are catalytic RNA

molecules that function

as enzymes and can

splice RNA

Figure 17.11

RNA transcript (pre-mRNA)

Exon 1 Intron Exon 2

Other proteins

Protein

snRNA

snRNPs

Spliceosome

Spliceosome

components

Cut-out

intronmRNA

Exon 1 Exon 2

5

5

5

1

2

3

The Functional and Evolutionary Importance of


51/73

y p

Introns

The presence of introns Allows for alternative RNA splicing

Variable processing of exons creates a family of proteins, exp.Antibodies, neurotrasmitter

F t t i


52/73

mRNA

From gene to protein

DNAtranscription

nucleuscytoplasm

mRNA leavesnucleus throughnuclear pores

proteins synthesized

by ribosomes usinginstructions on mRNA

a

a

a

a

a

a

a

a

a

a

a

a

a

a

aa

ribosome

proteintranslation

H d RNA d f t i ?


53/73

How does mRNA code for proteins?

TACGCACATTTACGTACGCGGDNA

AUGCGUGUAAAUGCAUGCGCCmRNA

MetArgValAsnAlaCysAlaprotein

?

How can you code for 20 amino acids

with only 4 nucleotide bases (A,U,G,C)?

C ki th d


54/73

Cracking the code

Nirenberg & Matthaei determined 1st codonamino acid match

UUU coded forphenylalanine

created artificial poly(U) mRNA

added mRNA to test tube of

ribosomes, tRNA & amino acids

mRNA synthesized single

amino acid polypeptide chain

1960 | 1968

phephephephephephe

How are the codons matched to amino


55/73

acids?

TACGCACATTTACGTACGCGGDNA

AUGCGUGUAAAUGCAUGCGCCmRNA

aminoacid

tRNA

anti-codon

codon

5' 3'

3' 5'

3' 5'

UAC

Met GCA

ArgCAU

Val


56/73


57/73

tRNA structure


58/73

tRNA structure

Clover leaf structure

anticodon on clover leaf end and amino acid attached on

3' end

consists of a single RNA strand that is only about 80

nucleotides long

Loading tRNA


59/73

Loading tRNA

Aminoacyl tRNAsynthetase

enzyme which bondsamino acid to tRNA

endergonic reaction ATP AMP

energy stored intRNA-amino acid bond

unstable

so it can release aminoacid at ribosome

Ribosomes


60/73

Ribosomes

Function protein production

Structure

ribosomes contain rRNA & protein

composed of 2 subunits that combine to carryout protein synthesis

P k t k t ib


61/73

Prokaryote vs. eukaryote ribosomes

Ribosomes


62/73

Ribosomes

Facilitate coupling oftRNA anticodon tomRNA codon

P site (peptidyl-tRNA site) holds tRNA carrying growing

polypeptide chain A site (aminoacyl-tRNA site)

holds tRNA carrying nextamino acid to be added tochain

E site (exit site) empty tRNAleaves ribosome

from exit site

Translation


63/73

Translation

Translation Is the actual synthesis of a

polypeptide, which occursunder the direction ofmRNA

Occurs on ribosomes

Codons blocks of 3 nucleotides

decoded intothe sequence of aminoacids

Codons must be read inthe correct reading frame For the specified

polypeptide to be produced

MEKANISME TRANSLASI


64/73

MEKANISME TRANSLASI

= aktivator, o=operator, p=promotor,tsp=situs inisiasi transkripsi, RBS=ribosom

binding site, ATG=kodon start, STOP=kodonstop, t=terminator

Komponen translasi : kodon start, RBS(ribosom binding site) dan kodon stop

TRANSLASI


65/73

TRANSLASI

Translasi berlangsung dari aa pada ujung N(amino), berakhir di ujung C (karboksil)

Cetakan : mRNA, ditranslasi dari 5 ke 3

tRNA dan rRNA tidak ditranslasi, tetapi

berfungsi langsung

RBS pada mRNA berikatan dengan ribosom (di

sitoplasma)

Building a polypeptide


66/73

Building a polypeptide

Initiation brings together mRNA,

ribosome subunits,proteins (initiation factors) &initiator tRNA

Elongation

Termination

Elongation: growing a polypeptide


67/73

Elongation: growing a polypeptide

catalyzed by peptidyl

transferase

peptydil tRNA


68/73

Translation in Prokaryotes


69/73


Transcription & translation are simultaneous in

bacteria

DNA is in

cytoplasm

no mRNA

editing needed



70/73


Polyribosomes : anumber of ribosomes

can translate a single

mRNA molecule

simultaneously

Figure 17.20a, b

Growing

polypeptides

Completed

polypeptide

Incoming

ribosomal

subunits

Start of

mRNA

(5 end)

End of

mRNA

(3 end)

An mRNA molecule is generally translated simultaneously

by several ribosomes in clusters called polyribosomes.

(a)

Ribosomes

mRNA

This micrograph shows a large polyribosome in a prokaryoticcell (TEM).

0.1 m

(b)

Translation in Eukaryotes


71/73

Translation in Eukaryotes

In a eukaryotic cell The nuclear

envelope separates

transcription from

translation

Extensive RNA

processing occurs in

the nucleus

Protein Folding and Post-TranslationalM difi ti


72/73

Modifications

After translation : Proteins may be modified in ways that affect

their three-dimensional shape

Proteins destined for the endomembrane

system or for secretion

Must be transported into the ER

Have signal peptides to which a signal-

recognition particle (SRP) binds, enabling the

translation ribosome to bind to the ER

The signal mechanism for targeting proteins to the ER


73/73

Ribosome

mRNA

Signal

peptide

Signal-

recognition

particle

(SRP) SRPreceptor

protein

Translocation

complex

CYTOSOL

Signal

peptide

removed

ER

membrane

Protein

ERLUMEN

g g g p

Polypeptide

synthesis begins

on a free

ribosome in

the cytosol.

1 An SRP binds

to the signal

peptide, halting

synthesis

momentarily.

2 The SRP binds to a

receptor protein in the ER

membrane. This receptor

is part of a protein complex

(a translocation complex)

that has a membrane pore

and a signal-cleaving enzyme.

3 The SRP leaves, and

the polypeptide resumes

growing, meanwhile

translocating across the

membrane. (The signal

peptide stays attached

to the membrane.)

4 The signal-

cleaving

enzyme

cuts off the

signal peptide.

5 The rest of

the completed

polypeptide leaves

the ribosome and

folds into its final

conformation.

6

3. DNA, RNA and Protein Synthesis

Documents

Transcript of 3. DNA, RNA and Protein Synthesis