Lecture 14

DNA replication and

Transcription
Regulation of DNA Replication

Initiation

Elongation

Termination
Replication termination in E. col is sequence specific

Ter sites

After a circular DNA molecule is replicated in E.coli, the

resulting complete daughter DNA molecules remain
linked to each other. Type II DNA topoisomerases can
efficiently separate (or decatenate) these DNA circles.
 Replication termination in eukaryotes

Steps in eukaryotic DNA replication. Generic illustration of replication initiation (parts a,b), elongation (parts c,d) and five
events that are unique to replication termination (parts d–g). The replicative DNA helicase is depicted without reference
to a specific translocation mechanism; RNA primers are shown in dark blue. The order of the termination events is
hypothetical. Topo, topoisomerase. doi:10.1038/nrm.2017.42
MEDICAL CONNECTIONS
 Anti-cancer strategy targeting DNA Replication
Inhibition of replication initiation
Helicase loading Helicase activation

Initiation Inhibitors against

Mcm2-7 or DDK

Elongation

Termination
 Anti-cancer strategy targeting DNA Replication

Initiation

Elongation

Termination
Substrates required for DNA Synthesis:

1. Deoxynucleoside Triphosphates
2. a Primer:Template Junction
 MEDICAL CONNECTIONS
The central role of DNA replication during cell division makes it a common target for chemotherapeutic
drugs that aim to prevent the growth of tumors. These drugs target DNA replication at various stages.

5-fluorouracil (5-FU)
Pyrimidine analog

6-mercaptopurine (6-MP)
Purine analog

Inhibit the synthesis of dNTP nucleotides

5-FU is the major agent used in the treatment of colorectal cancer and is also used in the treatment
of stomach, pancreatic, and advanced breast cancer.

6-MP is primarily used to treat patients with acute leukemia (blood cell cancers).
 MEDICAL CONNECTIONS

Cytosine arabinoside (AraC):

deoxycytidine analog
after conversion to a nucleoside
triphosphate is incorporated into
DNA in place of dCTP

Once incorporated into the DNA, the difference between the deoxyribose sugar of dCTP and the
arabinose sugar of AraCTP leads to incorrect positioning of the 3’ -hydroxyl of the primer DNA and
termination of elongation.

AraC is primarily used in the treatment of acute leukemia.

Transcription
 Flow of Genetic Information

1958

reverse transcription RNA->RNA

Reverse transcription
RNA Polymerase
Polymerase

in the words of Francis Crick:

Once information has passed into protein, it
cannot get out again.
 RNA molecules are single-stranded
The chemical structure of RNA

Uracil forms base pairs with adenine

Like DNA, RNA is a linear polymer made of four different types of nucleotide subunits linked together by
phosphodiester bonds.
It differs from DNA chemically in two respects:
(1) the nucleotides in RNA are ribonucleotides— that is, they contain the sugar ribose (hence the name
A short length of RNA. ribonucleic acid) rather than deoxyribose;
(2) although, like DNA, RNA contains the bases adenine (A), guanine (G), and cytosine (C), it contains the base
uracil (U) instead of the thymine (T) in DNA.
 Double-stranded vs single-stranded

Whereas DNA always occurs in cells as a double-stranded helix, RNA is single-stranded.

An RNA chain can therefore fold up into a particular shape. The ability to fold into complex three-
dimensional shapes allows some RNA molecules to have precise structural and catalytic functions.
 RNA can fold into specific structures

Structure of an actual RNA

RNA is largely single-stranded, but it often contains short stretches of nucleotides that can form conventional base pairs
with complementary sequences found elsewhere on the same molecule. These interactions, along with additional
“nonconventional” base-pair interactions, allow an RNA molecule to fold into a three-dimensional structure that is
determined by its sequence of nucleotides.
(A) Diagram of a folded RNA structure showing only conventional base-pair interactions.
(B) Structure with both conventional (red) and nonconventional (green) base-pair interactions.
 RNA helices often contain nonconventional base pairs

conventional

nonconventional
 RNA polymerases
Comparison of the crystal structures of prokaryotic and eukaryotic RNA polymerases

Overall shape
Prokaryotic RP (Bacteria) Eukaryotic RP II (Yeast)
resembles a crab claw
 Bacterial (prokaryotic) cells have a single RNA
polymerase that makes all three types of RNA in the cell

RNA molecules in E. coli

Type Abbreviation Function Size in nucleotides

Transfer RNA tRNA Carries activated amino acid about 75

5S rRNA Ribosome component 120

Ribosomal RNA 16S rRNA Ribosome component 1542
23S rRNA Ribosome component 2904

Messenger RNA mRNA Codes for proteins variable

 Eukaryotic RNA polymerases

Eukaryotic RNA polymerases

Type Transcribes Location
RNA polymerase I Large rRNAs Nucleolus
RNA polymerase II Pre-mRNAs Nucleoplasm
RNA polymerase III tRNAs, small 5S rRNA, small nuclear RNAs Nucleoplasm
Three phases of the
transcription cycle

Initiation

Elongation

Termination
 Initiation

Elongation

Termination
 Signals Encoded in DNA Tell RNA Polymerase Where to
Start and Stop.

Prokaryotic Gene Structure

Polycistronic mRNA – An mRNA bearing information from
more than one gene
 DNA Sequence & Structure

Polycistronic mRNA

operon = a cluster of genes under the control of a single promoter

• Coding – gene
• Regulatory – e.g. promoter
 Strand Name
= Coding strand = Same sequence as mRNA

= Template strand

Transcription proceeds from 5’ --> 3’

 Genes can be expressed with different efficiencies

In this example, gene A is transcribed much more efficiently than gene B and each RNA molecule
that it produces is also translated more frequently. This causes the amount of protein A in the cell
to be much greater than that of protein B.
 Strong E.coli promotors share similar features

TATA box

• -35 to -10 core promoter elements upstream of coding sequence

• Determine transcription start site
• More similar to the consensus sequence, the “stronger” the promoter (the more efficient the initiation and escape)
Question
Which gene is more strongly transcribed?
• Consensus promoter: TTGACAT TATAAT
• Gene A promoter: TTTACAG TATGAT
• Gene B promoter: TTGACAA TATAAT
• Gene C promoter: TAGATAA TATCAT
 RNA polymerase core enzyme binding to the promoter in bacteria

𝛔 and 𝛂 subunits recruit

RNAP core to the promoter

The carboxy-terminal domain of the 𝛂 subunit (𝛂 CTD) recognizes the UP-element (upstream promoter
element), whereas 𝛔 regions 2 and 4 recognize the –10 and –35 regions, respectively.
This representation of RNA polymerase is particularly useful for indicating surfaces that touch DNA and
regulatory proteins.
 How to determine the precise DNA sequence to which RNAP binds

DNaseI Footprinting
Different occupancy of 𝛂2 factor
and RNAP at promoter region
The transcription cycle of
bacterial RNAP:
Initiation, Elongation,
Termination
In step 1, the RNA polymerase
holoenzyme (polymerase core enzyme
plus σ factor) assembles and then
locates a promoter DNA sequence.
The polymerase opens (unwinds) the DNA at the
position at which transcription is to begin (step 2)
Step 3: The polymerase begins transcribing.

This initial RNA synthesis (abortive initiation)

is relatively inefficient as short, unproductive
transcripts are often released.
Step 4:
Once RNA polymerase has managed to
synthesize about 10 nucleotides of RNA, it
breaks its interactions with the promoter DNA
Step 5:
Eventually RNAP releases σ factor as
the polymerase tightens around the
DNA and shifts to the elongation
mode of RNA synthesis, moving along
the DNA.

During the elongation mode,

transcription is highly processive.
During elongation, RNA polymerase:
• unwinds DNA in front,
• re-anneals it behind,
• dissociates RNA from DNA template
• proofreading
RNA polymerase leaves the DNA
template and releases the newly
transcribed RNA only when it
encounters a termination signal.

Termination signals are typically

encoded in DNA, and many
function by forming an RNA
hairpin-like structure that
destabilizes the polymerase’s hold
on the RNA.
Comparison of DNA Replication and RNA Transcription
DNA Replication RNA Transcription
Enzyme DNA polymerase RNA polymerase
Template DNA – only 1 strand
DNA – both strands
as template
Primer RNA de novo synthesis;
does not need primer
Product RNA – not base-paired
DNA; with template DNA,
double-stranded; but is displaced;
only being single-stranded
duplicated once per Multiple copies of
cell cycle transcripts from a
single gene
Accuracy Error 1/10,000,000,000 Error 1/10,000
Summary
1. Comparison of DNA Replication and RNA Transcription

2. RNA structure

3. RNA polymerases

4. Transcription in prokaryotes

a. DNA sequence and structure

b. Promotor
c. Transcription cycle: initiation, elongation, and termination