Nucleic Acid

The Central
Dogma

Replication

DNA
Transcription

RNA
Translation

protein
The flow of genetic information is unidirectional, from
DNA to protein with messenger RNA as an intermediate.
 DNA Replication
- the process of making new copies of the DNA molecules

Potential mechanisms:
organization of DNA strands

Conservative old/old + new/new

Semiconservative old/new + new/old

Dispersive mixed old and new on each

strand
Meselson and Stahl’s replication experiment
 Replication as a process
1. Double-stranded DNA unwinds.

2. The junction of the unwound

molecules is a replication fork.

3. A new strand is formed by pairing

complementary bases with the
old strand.

4. Two molecules are made.

Each has one new and one old
DNA strand.
 Enzymes in DNA replication

Helicase unwinds Binding proteins Primase adds

parental double helix stabilize separate short primer
strands to template strand

DNA polymerase Exonuclease removes Ligase joins Okazaki

binds nucleotides RNA primer and inserts fragments and seals
to form new strands the correct bases other nicks in sugar-
phosphate backbone
 Replication
3’
3’ 5’
5’
3’

5’ 3’
5’

Helicase protein binds to DNA sequences called

origins and unwinds DNA strands.
Binding proteins prevent single strands from rewinding.
Primase protein makes a short segment of RNA
complementary to the DNA, a primer.
 Replication
Overall direction 3’
of replication
3’ 5’
5’

3’

5’ 3’
5’

DNA polymerase enzyme adds DNA nucleotides

to the RNA primer.
 Replication
Overall direction
3’
of replication
3’ 5’

5’
3’

5’ 3’
5’

Leading strand synthesis continues in a

5’ to 3’ direction.
 Replication
Overall direction
3’
of replication
3’ 5’

5’
Okazaki fragment
3’

5’ 3’ 5’ 3’
5’

Leading strand synthesis continues in a

5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
 Replication
Overall direction
3’
of replication
3’ 5’

5’
Okazaki fragment
3’

5’ 3’ 5’ 3’
5’

Leading strand synthesis continues in a

5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
 Replication

3’
3’ 5’

5’
3’
5’ 3’ 5’ 3’5’ 3’
5’

Leading strand synthesis continues in a

5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
 Replication

3’
3’ 5’

5’
3’
5’ 3’5’ 3’5’ 3’
5’

Leading strand synthesis continues in a

5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.
 Replication

3’
3’ 5’

5’
3’
5’ 3’5’ 3’5’ 3’
5’

Exonuclease enzymes remove RNA primers.

 Replication

3’
3’

5’
3’
5’ 3’5’ 3’
5’

Exonuclease enzymes remove RNA primers.

Ligase forms bonds between sugar-phosphate

backbone.
 Replication
3’
3’ 5’ 3’ 5’
5’ 5’
3’ 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’
5’

3’
3’ 5’
5’
3’ 3’
5’ 3’ 3’ 5’
5’ 5’
3’ 5’ 3’ 5’ 3’
3’ 3’
3’ 5’ 5’
5’
3’ 5’ 3’
5’ 3’
3’
5’
3’ 3’
3’ 5’ 5’ 3’ 5’
5’ 3’
3’ 5’ 3’ 5’ 3’ 5’
5’
Transcription
 Terminology
• Only one strand of DNA serves as a
template for transcription.

• Different genes are transcribed from

different strands
 Prokaryotic Gene Structure
Promoter CDS Terminator
UTR UTR

Genomic DNA

transcription

mRNA

translation

protein
 Eukaryotic Gene Structure
5’ - Promoter Exon1 Intron1 Exon2 Terminator – 3’
UTR splice splice UTR

transcription
Poly A

translation

protein
 Promoter
• Promoter determines:

1. Which strand will serve as a template.

2. Transcription starting point.

3. Strength of polymerase binding.

4. Frequency of polymerase binding.

 Prokaryotic Promoter

• One type of RNA polymerase.

• Pribnow box located at –10 (6-7bp)
• –35 sequence located at -35 (6bp)
 Promoter region of E. coli genes bound
by RNA pol. containing s70
Promoter region
RNA polymerase binding
-35 region Pribnow-box Transcriptional Translational
“RNA pol. TATA-box start site start site
recognition site” -10 region -1 +1 ~+40
5’-TTGACA— 16-18 —TATAAT— 6-8 — NAC--NATG...– 3’
3’-AACTGT— 16-18 —ATATTA— 6-8 — NTG--NTAC...– 5’

Consensus
(average) seq. for
the – 35 region
pppAC--NAUG...– 3’
Met...
Begin of RNA transcript
Begin of coding sequence
 Promoters Strength
– Weak promoters:
• low degree of similarity with consensus TATA-box and the -
35 region, or spacing b/n them is not 17 nts
• transcribed about once every 10 min. in E. coli
– Strong promoters:
• high degree of identity with TATA box & -35 region
• may be transcribed as high as every 2 sec. in E. coli
• Other factors
 Eukaryote Promoter

• 3 types of RNA polymerases are employed

in transcription of genes:
– RNA polymerase I transcribes rRNA
– RNA polymerase II transcribes all genes coding
for polypeptides
– RNA polymerase III transcribes small
cytoplasmatic RNA, such as tRNA.
 Eukaryote Promoter
• Goldberg-Hogness or TATA located at –30
• Additional regions at –100 and at –200
• Possible distant regions acting as enhancers
or silencers (even more than 50 kb).
 Promoter sequences recognized
by RNA polymerase II
- 40 to - 150 - 30 to -100 +1
TATAAA YYANWYY
Regulatory TATA-box Inr
sequences Initiator sequence

Examples

Also effective on the

GGNCAATCT GGGCGG template strand.
CAAT-box GC-box Recognized by
(constitutive genes) transcription factors
 RNA Synthesis
 DNA template: 3’-to-5’
 RNA synthesis: 5’-3’; no primer needed

30
Transcription initiation and elongation

1. Genes need to be
expressed to be
“genes”

2. Transcription is
directed to specific
locations
(promoters)

3. RNA is elongated in
the 5’-to-3’
direction
 Termination Sites

• The newly synthesized mRNA forms a stem

and loop structure (lollipop).
• A disassociation signal at the end of the
gene that stops elongating and releases
RNA polymerase.
• All terminators (eukaryotes and
prokaryotes) form a secondary structure.
 Termination Sites
• The terminator region pauses the
polymerase and causes disassociation.
 Rho-independent termination
Termination signal (example)

- Palindromic GC-rich region

followed by an AT-rich region

- The transcripts usually ends

shortly after the hairpin

- RNA polymerase pauses after

the newly synthesized stretch of
mRNA forms a hairpin

Source: Stryer pg. 788

 Rho-dependent termination
Rho-factor
- hexameric protein
- Trails RNA polymerase 5’-3’
and hydrolyzes ATP when
ssRNA is present
- Rho is activated by
sequences in the nascent
mRNA molecule that are
rich in cytosine and poor in
guanine
- Rho then displaces the
RNA polymerase from the
DNA template (RNA-DNA
helicase)
 Splice Sites
• Eukaryotics only
• Removing internal parts of the newly transcribed
RNA.
• Takes place in the cell nucleus (hnRNA)
Translation
From Gene to Protein
Open Reading Frames (ORF)
 The Codon
• mRNA sequence is decoded in sets of three
nucleotides.
• Since there are 64 possible tri-nucleotide
combinations and only 20 amino acids, there must
be some redundancy (a.k.a degenerate code).
 tRNA Needed

• tRNA or transfer RNA is needed for translating the

codon to amino acid sequence.
• The tRNA is linked to an amino acid by an amino-
acyl tRNA synthetase.
• There are two tRNAs for Methionine, of which only
the initiation-specific tRNA can be used to start
translation.
tRNA
Amino acid attachment to tRNA
codon - anticodon
 Wobble

• Due to “non-standard” interactions, some tRNAs can base-

pair with just two complementary bases.
• The third position can be mismatched (wobble).
• This allows the 61 codons to be matched by as few as 31
different tRNAs.
• In part, this can be due to non-standard bases, such as
inosine, which is de-aminated adenine and can base-pair
with A, C, and U. In addition, G can basepair with U,
although its normal partner is C.
Wobble hypothesis:

 Proposed by Francis Crick in 1966.

 Occurs at 3’ end of codon/5’ end of anti-codon.

 Result of arrangement of H-bonds of base pairs at the 3rd pos.

 Degeneracy of the code is such that wobble always results in

translation of the same amino acid.

 Complete set of codons can be read by fewer than 61 tRNAs.

5’ anti-codon 3’ codon

G pairs with U or C
C pairs with G
A pairs with U
U pairs with A or G
I (Inosine) pairs with A, U, or C
I = post-transcription modified purine

Fig. 6.9
 All possible base pairings at the wobble
position
No purine-purine or pyrimidine-pyrimidine
base pairs are allowed as ribose distances
would be incorrect (Neat!).
 Start Methionine

AUG is start signal for most proteins,

specifying an N-terminal methionine
Ribosome
Formation of initiation complex
Termination

Two kinds of termination factors:

• Recognize STOP codons
• Hydrolyze peptidyl-tRNA bond
 Translation Summary
• mRNA leaves the nucleus via nuclear pores.
• Ribosome has 3 binding sites for tRNAs:
– A-site: position that aminoacyl-tRNA
molecule binds to vacant site
– P-site: site where the new peptide bond
is formed.
– E-site: the exit site
• Two subunits join together on a mRNA
molecule near the 5’ end.
• The ribosome will read the codons until
AUG is reached and then the initiator tRNA
binds to the P-site of the ribosome.
• Stop codons have tRNA that recognize a
signal to stop translation. Release factors
bind to the ribosome which cause the
peptidyl transferase to catalyze the addition
of water to free the molecule and releases
the polypeptide.
See you later。。。。。。

See You Later …….