Lecture 15

Transcription 2
 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
The overall structure of the E. coli genome

Protein-coding genes account for 87.8% of

the genome, 0.8% encodes stable RNAs, and
0.7% consists of noncoding repeats, leaving
∼11% for regulatory and other functions.

The origin and terminus of replication are shown as green

lines, with blue arrows indicating replichores 1 and 2.

The distribution of genes is depicted on two outer rings:

The orange boxes are genes located on the presented
strand, and the yellow boxes are genes on the opposite
strand.

Red arrows show the location and direction of

transcription of rRNA genes, and tRNA genes are shown
as green arrows.

Science 05 Sep 1997:

Vol. 277, Issue 5331, pp. 1453-1462
DOI: 10.1126/science.277.5331.1453
 Operons: Fine Control of Bacterial Transcription

The E. coli genome contains over 3000 genes. Some of these are active all the time
because their products are in constant demand. But some of them are turned off
most of the time because their products are rarely needed.
The lac Operon

Diauxic growth.

E. coli cells are grown on a medium

containing both glucose and lactose, and
the bacterial density (number of cells/mL)
is plotted versus time in hours.

The cells grow rapidly on glucose until that

sugar is exhausted, then growth levels off
while the cells induce the enzymes needed
to metabolize lactose. As those enzymes
appear, growth resumes.
 What are these enzymes?

First, the bacteria need an enzyme to transport

the lactose into the cells. The name of this
galactoside permease enzyme is galactoside permease.

Next, the cells need an enzyme to break the

lactose down into its two component sugars:
galactose and glucose.

Because lactose is composed of two simple

sugars, we call it a disaccharide. These six-
𝛽-galactosidase carbon sugars, galactose and glucose, are joined
together by a linkage called a 𝛽-galactosidic
bond. Lactose is therefore called a 𝛽-
galactoside, and the enzyme that cuts it in half is
called 𝛽-galactosidase.
 The lac Operon
glucose lactose

The three genes coding for enzymes that carry out lactose metabolism are grouped together in the
following order:
𝛽 -galactosidase (lacZ)
galactoside permease (lacY)
galactoside transacetylase (lacA)--structural gene whose function in lactose
metabolism is still unclear
They are all transcribed together to produce one messenger RNA, called a polycistronic message,
starting from a single promoter.
 The lac Operon

CAP: catabolite activator protein The CAP site and the operator
(the site bound by Lac repressor)
are each about 20 bp. The
operator lies within the region
bound by RNA polymerase at the
promoter, and the CAP site lies
just upstream of the promoter.

The control region of the lac operon

Expression of the lac genes at three levels

The presence or absence of the sugars

lactose and glucose control the level of
expression of the lac genes.

High levels of expression require the

presence of lactose (and hence the absence
of functional Lac repressor) and absence of
the preferred energy source, glucose (and
OFF
hence presence of the activator CAP).

When bound to the operator, Lac repressor

OFF excludes polymerase whether or not active
CAP is present. CAP and Lac repressor are
shown as single units, but CAP actually binds
DNA as a dimer, and Lac repressor binds as a
tetramer. CAP recruits polymerase to the lac
ON promoter where it spontaneously undergoes
isomerization to the open complex (the state
shown in the bottom line).
 How does repressor work?

negative control

Negative control, which is like the brake of a car: You need to release the brake for the car
to move. The “brake” in negative control is a protein called the lac repressor, which keeps
the operon turned off (or repressed) as long as lactose is absent. That is economical; it
would be wasteful for the cell to produce enzymes that use an absent sugar.
 The lac suppressor
•An N-terminal DNA-binding domain (in which two LacI
N proteins bind a single operator site)
•A regulatory domain (sometimes called the core domain,
which binds allolactose, an allosteric effector molecule)
•A linker that connects the DNA-binding domain with the core
domain (sometimes called the hinge helix, which is important
for allosteric communication)
•A C-terminal tetramerization region (which joins four
monomers in an alpha-helix bundle)

C
Looped non-
coding DNA
N Genes

Lactose-Repressor
C Proteins
 The inducer of the lac operon is allolactose

lacto sidase
𝛽-ga

When lactose enters the cell, it is converted to allolactose. It is allolactose (rather than lactose itself ) that controls
the Lac repressor. Paradoxically, the conversion of lactose to allolactose is catalyzed by 𝛽-galactosidase, itself
encoded by one of the lac genes. How is this possible?
The answer is that expression of the lac genes is leaky: even when they are repressed, an occasional transcript gets
made. This happens because every so often, RNA polymerase will manage to bind the promoter in place of the Lac
repressor. This leakiness ensures that there is a low level of 𝛽-galactosidase in the cell even in the absence of
lactose, and so there is enzyme poised to catalyze the conversion of lactose to allolactose.
The application of 𝛽-galactosidase in biological research

𝛽-galactosidase

X-gal assay Analog of inducer: IPTG

 X-gal assay
Blue-white screen

𝛽-galactosidase

X-gal assay

The blue–white screen is a screening technique that allows for the

rapid and convenient detection of recombinant bacteria in vector-
based molecular cloning experiments. DNA of interest is ligated into
a vector. The vector is then inserted into a competent host cell
viable for transformation, which are then grown in the presence
of X-gal. Cells transformed with vectors containing recombinant DNA
will produce white colonies; cells transformed with non-
recombinant plasmids (i.e. only the vector) grow into blue colonies..
IPTG for inducing protein expression
Allolactose binds to the Lac repressor and triggers a
change in the shape (conformation) of that protein

floppy

- allolactose + allolactose
 An Activator and a Repressor Together Control the lac Genes

CAP: catabolite activator protein

Activation of the lac promoter by CAP

CAP binding site is located some 60 bp upstream

of the start site of transcription. When CAP binds
to that site, the activator helps polymerase bind
to the promoter by interacting with the enzyme
RNA polymerase binds the lac promoter poorly in the and recruiting it to the promoter. This
absence of CAP, even when there is no functional cooperative binding stabilizes the binding of
repressor present. polymerase to the promoter.
 Structure of CAP– 𝛼CTD–DNA complex

𝛼-CTD
cAMP
CAP dimer

CAP is shown in cyan bound as a dimer to its site on DNA. In addition, the 𝛼-CTD of RNA polymerase
is shown (in purple) bound to an adjacent stretch of DNA and interacting with CAP. The site of
interaction on each protein involves the residues identified genetically. One molecule of cAMP is
shown bound to each monomer of CAP.
 Relationship between glucose and cAMP

êGlucose è é cAMP

http://www.discoveryandinnovation.com/BIOL202/notes/lecture16.html
 Mechanism of allosteric control of CAP
The crystal structures of CAP in three states have been determined.

CAP alone CAP bound to cAMP CAP–cAMP–DNA

From these it is clear how binding of cAMP (to the cAMP binding domains [CBDs] of CAP) causes a structural change in
the protein that results in its DNA-binding domains being realigned to an optimum configuration for DNA recognition.
This happens because cAMP bound to CBDs also makes hydrogen bonds to two residues in a coiled region of the
protein, triggering that region to form a helix (shown in blue). The recognition helices of CAP’s DNA-binding domain
are shown in orange.
Summary

1. Diauxic growth

2. The lac Operon

3. Expression of the lac genes at three levels

4. Suppressor-Lac I

5. Inducer-allolactose
X-gal, IPTG

6. Activator-CAP