3.

DNA, RNA and Protein Synthesis

LOGO

Where it all began

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

.

And now look at you

How did you get from there to here?

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

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

DNA Replication
The double helix unwinds into 2 separate strands. The two strands serve as templates for copying two new strands of DNA each new helix contains 1 original strand plus 1 new strand semiconservative 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 to make the DNA.

Nucleotide precursors ATP Adenosine triphosphate

modified nucleotide
adenine ribose + Pi + Pi + Pi + +

Bacterial chromosome replication

The separation of complementary strands occurs in the regions of the DNA called origins of replication (ori). Bacterial chromosome have a single origin. The separated strands form replication fork

Eukaryotic chromosome replication

Because of their large size, eukaryotic chromosome have multiple origins
Origin of replication Parental (template) strand Daughter (new) strand 0.25 m

1 Replication begins at specific sites where the two parental strands separate and form replication bubbles.
2 The bubbles expand laterally, as DNA replication proceeds in both directions. 3 Eventually, the replication bubbles fuse, and synthesis of the daughter strands is complete.

Bubble

Replication fork

Two daughter DNA molecules

(a) In eukaryotes, DNA replication begins at many sites along the giant DNA molecule of each chromosome.

(b) In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM).

DNA Replication
Addition of RNA primer (10-15 bp), are synthesized by primase

DNA gyrase

Antiparallel Elongation
DNA polymerases cannot initiate the synthesis of a polynucleotide They can only add nucleotides to the 3 end (5 3) The initial nucleotide strand is a primer = RNA sequences, are synthesized by primase
5 5 RNA primer 3

5 newly synthesized DNA

Synthesis of the strands

RNA primer direction of leading strand synthesis 3 5 replication fork 5 3

3 5
direction of lagging strand synthesis

Synthesis of the strands

Movement of the replication fork

5 3

5 3

Synthesis of the strands

Movement of the replication fork

RNA primer Okazaki fragment RNA primer

RNA primer 5

pol III 5 DNA polymerase III initiates at the primer and elongates DNA up to the next RNA primer

newly synthesized DNA (100-1000 bases) (Okazaki fragment) 5

pol I

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

Synthesis of the strands

newly synthesized DNA (Okazaki fragment)

DNA ligase seals the gap by catalyzing the formation of a 3, 5-phosphodiester bond in an ATP-dependent reaction 5

Strand separation at the replication fork causes positive supercoiling of the downstream double helix

3 5 5 3 3 5 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)

A summary of DNA replication

Overall direction of replication

1 Helicase unwinds the parental double helix.

2 Molecules of singlestrand binding protein stabilize the unwound template strands.
3 The leading strand is synthesized continuously in the 5 3 direction by DNA pol III.

Lagging Leading strand Origin of replication strand

DNA pol III

Lagging strand Leading strand

OVERVIEW

Leading strand

5 3 Parental DNA 4 Primase begins synthesis of RNA primer for fifth Okazaki fragment.

Replication fork Primase DNA pol III Primer 4

DNA pol I Lagging strand 3 2

DNA ligase 1 3

5 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.

6 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 sugarphosphate backbone with a free 3 end.

7 DNA ligase bonds the 3 end of the second fragment to the 5 end of the first fragment.

Figure 16.16

Proteins That Assist DNA Replication

Table 16.1

Proteins That Assist DNA Replication : DNA Polymerase

Prokaryote Pol I Pol II Pol III Function Gap filling & DNA repair Gap filling & DNA repair Replication

Eukaryote Pol
Pol Pol

Lagging strand replication

Gap filling & DNA repair Leading strand replication

Pol
Pol

Mitochondria replication
Gap filling & DNA repair

Replicating the Ends of DNA Molecules

Eukaryotic chromosomal DNA molecules
Have at their ends nucleotide sequences, called telomeres, that postpone the erosion of genes near the ends of DNA molecules

If the chromosomes of germ cells became shorter 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

The ends of eukaryotic chromosomal DNA
Get shorter with each round of replication
5 End of parental DNA strands 3 Leading strand Lagging strand

Last fragment RNA primer Lagging strand

Previous fragment

5
3

An enzyme called telomerase

Catalyzes the lengthening of telomeres in germ cells

Primer removed but cannot be replaced with DNA because no 3 end available for DNA polymerase 3

Removal of primers and replacement with DNA where a 3 end is available 5

Second round of replication

5
New leading strand 3 New lagging strand 5

3
Further rounds of replication

Figure 16.18

Shorter and shorter daughter molecules

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

Central Dogma
Replication

DNA
Reverse Transcription

Transcription

RNA
Translation

Protein

Juang RH (2004) BCbasics

RNA ribose sugar N-bases

uracil instead of thymine, which U : A modified bases

single stranded
transcription

DNA

RNA

RNA polynucleotide chain

2 -OH makes 3, 5 phosphodiester bond unstable

DNA polynucleotide chain

Examples of modified bases found in RNA

Dihydrouridine

Pseudouridine

1-methylguanosine

7-methylguanosine

1-methyladenosine

2-thiocytidine

5-methylcytidine

Ribothymine

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

Transcription
Transcription
Is the synthesis of RNA under the direction of DNA

Occurs only in a chromosome segments that contain 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 then copies one strand of DNA to RNA

Template (-) Strand Transcribes to mRNA

nontemplate strand

(+) strand

coding strand

5
ATG TAG
Identical sequence

Codon is denoted on mRNA

Start codon

AUG TAC

UAG ATC (-) strand

3 RNA 5

template strand

Juang RH (2004) BCbasics

Transcription

Transcription bubble

MEKANISME TRANSKRIPSI

= aktivator, o=operator, p=promotor, tsp=situs inisiasi transkripsi, RBS=ribosom binding site, ATG=kodon start, STOP=kodon stop, t=terminator Komponen transkripsi : promotor, situs inisiasi transkripsi, dan terminator

MEKANISME TRANSKRIPSI
Promotor : tempat RNA pol mengikatkan diri utk memulai 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 2 membuat blok 1 membuka mjd untai tunggal. Salah satu untai tunggal menjadi templat dan transkripsi dimulai di +1.

Transcription in Prokaryotes Promoter sequences

RNA polymerase molecules bound to bacterial DNA

Transcription in Prokaryotes Initiation

RNA polymerase binds to promoter sequence on DNA

Transcription in Prokaryotes 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

Transcription in Prokaryotes Termination

RNA polymerase stops at termination sequence mRNA leaves nucleus through pores

RNA GC hairpin turn

Structure of prokaryotic mRNA

Shine-Dalgarno sequence PuPuPuPuPuPuPuPu 3 AAU termination initiation AUG

translated region

The Shine-Dalgarno (SD) sequence base-pairs with a pyrimidine-rich sequence in 16S rRNA to facilitate the initiation of protein synthesis

Transcription in Eukaryotes

LOGO

Prokaryote vs Eukaryote genes

Prokaryotes
DNA in cytoplasm circular chromosome naked DNA

Eukaryotes
DNA in nucleus linear chromosomes DNA wound on histone proteins introns vs. exons

no introns

intron = noncoding (in between) sequence eukaryotic DNA exon = coding (expressed) sequence

Intron and Exon in Eukaryotic Cells

promotor exon intron exonintron exon

DNA
5
stop codon

start codon

Transcription

5
Processing

3 mRNA

cap
Splicing Intron deleted Take place in nucleus poly A tail

mature mRNA

To cytoplasm
Juang RH (2004) BCbasics

Transcription in Eukaryotes
Promoters signal the initiation of RNA synthesis Transcription factors
Help eukaryotic RNA polymerase recognize promoter sequences

Initiation complex
transcription factors bind to promoter region upstream of gene
proteins which bind to DNA & turn on or off transcription TATA box binding site

only then does RNA polymerase bind to DNA

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 activator proteins enhanced rate (high level) of transcription

Transcription in Eukaryotes 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

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 mRNA

from RNase enzymes in cytoplasm by alteration of mRNA ends add 5' cap add polyA tail
A modified guanine nucleotide added to the 5 end
TRANSCRIPTION DNA Pre-mRNA

50 to 250 adenine nucleotides added to the 3 end Polyadenylation signal 3 AAUAAA AAAAAA Poly-A tail

RNA PROCESSING mRNA

5 G P P P
Ribosome

Protein-coding segment

TRANSLATION

5 Cap
Polypeptide

5 UTR

Start codon Stop codon

3 UTR

Add 5 Cap

cap plays a role in ribosome recognition of the 5 end of the RNA molecule during translation

Polyadenylation
Polyadenylation : a string of adenine nucleotides around 100- 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 nucleotide added to the 5 end

TRANSCRIPTION DNA Pre-mRNA

50 to 250 adenine nucleotides added to the 3 end Polyadenylation signal 3 AAUAAA AAAAAA Poly-A tail

RNA PROCESSING mRNA

5 G P P P
Ribosome

Protein-coding segment

TRANSLATION

5 Cap
Polypeptide

5 UTR

Start codon Stop codon

3 UTR

Post-transcriptional processing RNA splicing

Removes introns and joins exons
TRANSCRIPTION DNA

RNA PROCESSING mRNA

Pre-mRNA

5 Exon Intron Pre-mRNA 5 Cap 30 31 1

Exon 104

Intron 105

Exon

3 Poly-A tail 146

Ribosome TRANSLATION

Coding segment mRNA 5 Cap 1 3 UTR

Introns cut out and exons spliced together Poly-A tail 146 3 UTR

Polypeptide

Figure 17.10

RNA splicing
Is carried out by spliceosomes 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 5 RNA transcript (pre-mRNA) Exon 1 Protein
1

Intron

Exon 2

snRNA snRNPs

Other proteins

Spliceosome

Spliceosome components

mRNA 5 Exon 1 Exon 2

Cut-out intron

The Functional and Evolutionary Importance of Introns

The presence of introns
Allows for alternative RNA splicing Variable processing of exons creates a family of proteins, exp. Antibodies, neurotrasmitter

From gene to protein

a a

transcription

translation

a a

a a

DNA

mRNA
a a ribosome

protein
a a a a

a a

a a

mRNA leaves nucleus through nuclear pores

nucleus

cytoplasm

proteins synthesized by ribosomes using instructions on mRNA

How does mRNA code for proteins?

DNA

TACGCACATTTACGTACGCGG

mRNA AUGCGUGUAAAUGCAUGCGCC

?
protein

Met ArgVal Asn Ala Cys Ala

How can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)?

Cracking the code Nirenberg & Matthaei

1960 | 1968

determined 1st codonamino acid match

UUU coded for phenylalanine

created artificial poly(U) mRNA added mRNA to test tube of ribosomes, tRNA & amino acids
mRNA synthesized single amino acid polypeptide chain

phephephephephephe

How are the codons matched to amino acids?

DNA

TACGCACATTTACGTACGCGG
5' 3'

3'

5'

mRNA AUGCGUGUAAAUGCAUGCGCC
codon
3' 5'

tRNA amino acid

UAC
Met

GCA
Arg

CAU anti-codon
Val

Codon-anticodon interactions
codon-anticodon base-pairing is antiparallel the third position in the codon is frequently degenerate one tRNA can interact with more than one codon (therefore 50 tRNAs) wobble rules C with G or I (inosine) 3 5 tRNAmet A with U or I G with C or U U with A, G, or I UAC I with C, U, or A

AUG 5

mRNA 3 5
GAU CUA G

tRNAleu
wobble base

one tRNAleu can read two of the leucine codons

mRNA

The code
For ALL life!
strongest support for a common origin for all life

Code is redundant
several codons for each amino acid

Why is this a good thing?

Start codon
AUG methionine

Stop codons
UGA, UAA, UAG

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 Aminoacyl tRNA synthetase

enzyme which bonds amino acid to tRNA endergonic reaction
ATP AMP

energy stored in tRNA-amino acid bond

unstable so it can release amino acid at ribosome

Ribosomes Function
protein production

Structure
ribosomes contain rRNA & protein composed of 2 subunits that combine to carry out protein synthesis

Prokaryote vs. eukaryote ribosomes

Ribosomes
Facilitate coupling of tRNA anticodon to mRNA codon P site (peptidyl-tRNA site)
holds tRNA carrying growing polypeptide chain

A site (aminoacyl-tRNA site)

holds tRNA carrying next amino acid to be added to chain

E site (exit site)

empty tRNAleaves ribosome from exit site

Translation
Translation
Is the actual synthesis of a polypeptide, which occurs under the direction of mRNA Occurs on ribosomes

Codons
blocks of 3 nucleotides decoded into the sequence of amino acids

Codons must be read in the correct reading frame

For the specified polypeptide to be produced

MEKANISME TRANSLASI

= aktivator, o=operator, p=promotor, tsp=situs inisiasi transkripsi, RBS=ribosom binding site, ATG=kodon start, STOP=kodon stop, t=terminator Komponen translasi : kodon start, RBS (ribosom binding site) dan kodon stop

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 Initiation

brings together mRNA, ribosome subunits, proteins (initiation factors) & initiator tRNA

Elongation Termination

Elongation: growing a polypeptide

peptydil tRNA

catalyzed by peptidyl transferase

Termination: release polypeptide Release factor

release protein bonds to A site bonds water molecule to polypeptide chain
Now what happens to the polypeptide?

Translation in Prokaryotes
Transcription & translation are simultaneous in bacteria DNA is in cytoplasm no mRNA editing needed

Translation in Prokaryotes
Polyribosomes : a number of ribosomes can translate a single mRNA molecule simultaneously
Growing polypeptides Incoming ribosomal subunits Start of mRNA (5 end) End of mRNA (3 end) Completed polypeptide

(a) An mRNA molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes.

Ribosomes mRNA

0.1 m

Figure 17.20a, b

(b) This micrograph shows a large polyribosome in a prokaryotic cell (TEM).

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-Translational 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 signalrecognition particle (SRP) binds, enabling the translation ribosome to bind to the ER

The signal mechanism for targeting proteins to the ER

1 Polypeptide synthesis begins on a free ribosome in the cytosol.

2 An SRP binds to the signal peptide, halting synthesis momentarily.

3 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.

4 The SRP leaves, and the polypeptide resumes growing, meanwhile translocating across the membrane. (The signal peptide stays attached to the membrane.)

5 The signalcleaving enzyme cuts off the signal peptide.

6 The rest of the completed polypeptide leaves the ribosome and folds into its final conformation.

Ribosome

mRNA Signal peptide Signalrecognition particle (SRP) SRP receptor CYTOSOL protein Translocation complex Signal peptide removed

ER membrane Protein

ERLUMEN

Figure 17.21