Lesson 3

Transcription in Bacteria
A gene participates in three major activities:

• 1. A gene is a repository of information. That is, it holds the

information for making one of the key molecules of life, an RNA. The
sequence of bases in the RNA depends directly on the sequence of
bases in the gene. Most of these RNAs, in turn, serve as templates for
making other critical cellular molecules, proteins. The production of
RNAs and proteins from a DNA blueprint is called gene expression.
• 2. A gene can be replicated. This duplication is very faithful, so the
genetic information can be passed essentially unchanged from
generation to generation.
• 3. A gene can accept occasional changes, or mutations. This allows
organisms to evolve. Sometimes, these changes involve
recombination, exchange of DNA between chromosomes or sites
within a chromosome. A subset of recombination events involve
pieces of DNA (transposable elements) that move from one place to
Storing Information
Overview of Gene Expressi
on

• DNA and protein have this

informational relationship:

Three nucleotides in the DNA gene

stand for one amino acid in a protein

• Producing a protein from information in a DNA gene is a two-step process. The first
step is synthesis of an RNA that is complementary to one of the strands of DNA. Th
is is called transcription. In the second step, called translation, the information in the
RNA is used to make a polypeptide. Such an informational RNA is called a messeng
er RNA (mRNA) to denote the fact that it carries information —like a message —fr
om a gene to the cell ’s protein factories.
• Outline of gene expression. In the first step, transcription, t
he template strand (black) is transcribed into mRNA. Note
that the non-template strand (blue) of the DNA has the sam
e sequence (except for the T –U change) as the mRNA (re
d). In the second step, the mRNA is translated into protein
(green). This little “gene ” is only 12 bp long and codes for
only four amino acids (a tetrapeptide). Real genes are muc
h larger.
• The above figure summarizes the process of expressing a protei
n-encoding gene and introduces the nomenclature we apply to th
e strands of DNA. Notice that the mRNA has the same sequence
(except that U ’s substitute for T ’s) as the top strand (blue) of th
e DNA. An mRNA holds the information for making a polypepti
de, so we say it “codes for ” a polypeptide, or “encodes ” a poly
peptide. ( Note: It is redundant to say “encodes for ” a polypepti
de.) In this case, the mRNA codes for the following string of am
ino acids: methionine-serine-asparagine-alanine, which is abbre
viated Met-Ser-Asn-Ala. We can see that the codeword (or codo
n ) for methionine in this mRNA is the triplet AUG; similarly, th
e codons for serine, asparagine, and alanine are AGU, AAC, and
GCG, respectively.
• Because the bottom DNA strand is complementary to the mRNA, w
e know that it served as the template for making the mRNA. Thus,
we call the bottom strand the template strand, or the transcribed s
trand. For the same reason, the top strand is the nontemplate stran
d, or the non transcribed strand. Because the top strand in our ex
ample has essentially the same coding properties as the correspondi
ng mRNA, many geneticists call it the coding strand. The opposite
strand would therefore be the anticoding strand. Also, since the top
strand has the same sense as the mRNA, this same system of nomen
clature refers to this top strand as the sense strand, and to the botto
m strand as the antisense strand. However, many other geneticists
use the “coding strand ” and “sense strand ” conventions in exactly
the opposite way. From now on, to avoid confusion, we will use the
unambiguous terms template strand and nontemplate strand
Protein Structure
• Because we are seeking to understand gene expression, and becaus
e proteins are the fi nal products of most genes, let us take a brief l
ook at the nature of proteins. Proteins, like nucleic acids, are chai
n-like polymers of small subunits. In the case of DNA and RNA, t
he links in the chain are nucleotides. The chain links of proteins ar
e amino acids. Whereas DNA contains only four different nucleoti
des, proteins contain 20 different amino acids.

• Each amino acid has an amino group (NH3 + ), a carboxyl group

(COO- ), a hydrogen atom (H), and a side chain. The only differenc
e between any two amino acids is in their different side chains. Th
us, it is the arrangement of amino acids, with their distinct side cha
ins, that gives each protein its unique character.
• Amino acid structure.
(a) The general
structure of an amino
acid. It has both an
amino group (NH3+ ;
red) and an acid
group (COO – ; blue);
hence the name. Its
other two positions
are occupied by a
hydrogen (H) and a
side chain (R, green).
(b) Each of the 20
different amino acids
has a different side
chain. All of them are
illustrated here.
Three-letter and one-
letter abbreviations
are in parentheses
 • The only difference b
etween any two amin
o acids is in their diffe
rent side chains. Thus,
it is the arrangement o
f amino acids, with th
eir distinct side chains
, that gives each prote
in its unique character
. The amino acids join
• A polypeptide chain has polarity, just as the DNA chain does. together in proteins vi
The dipeptide (two amino acids linked together) shown on the a peptide bonds ， Th
right in Figure 3.3 has a free amino group at its left end. This is gives rise to the na
is the amino terminus, or N-terminus. It also has a free me polypeptide for a
carboxyl group at its right end, which is the carboxyl chain of amino acids.
terminus, or C-terminus. The linear order of amino acids A protein can be com
constitutes a protein’s primary structure. The way these amino posed of one or more
acids interact with their neighbors gives a protein its polypeptides.
secondary structure.
• An example of protein secondary structure:
The a-helix. (a) The positions of the amino
acids in the helix are shown, with the helical
backbone in gray and blue. The dashed lines
represent hydrogen bonds between hydrogen
and oxygen atoms on nearby amino acids.
The small white circles represent hydrogen
atoms. (b) A simplified rendition of the a-
helix, showing only the atoms in the helical
backbone
• An antiparallel β-sheet. Two polypeptide chains a
re arranged side by side, with hydrogen bonds (da
shed lines) between them. The green and white pl
anes show that the β-sheet is pleated. The chains
are antiparallel in that the amino terminus of one
and the carboxyl terminus of the other are at the t
op. The arrows indicate that the two β-strands run
from amino to carboxyl terminal in opposite dire
ctions. Parallel β-sheets, in which the b-strands ru
n in the same direction, also exist.

• Silk is a protein very rich in β-pleated sheets. A t

hird example of secondary structure is simply a t
urn. Such turns connect the α-helices and β-pleat
ed sheet elements in a protein.
• The total three-d
imensional shap
e of a polypeptid
e is its tertiary st
ructure.

• Tertiary structure of myoglobin. The several a-

helical regions of this protein are represented by
turquoise corkscrews. The overall molecule seems
to resemble a sausage, twisted into a roughly
spherical or globular shape. The heme group is
shown in red, bound to two histidines (turquoise
polygons) in the protein.
• myoglobin is composed of
a single, more or less globul
ar, structure, but other prote
ins can contain more than o
ne compact structural regio
n. Each of these regions is c
alled a domain. domains ca
n contain common structura
l –functional motifs. Antibo
dies (the proteins that white
blood cells make to repel in
vaders) provide a good exa
mple of domains. Each of t
he four polypeptides in the I
gG-type antibody contains
globular domains
• the highest level of protein structure — quaternary structure —which is the w
ay two or more individual polypeptides fi t together in a complex protein. It h
as long been assumed that a protein ’s amino acid sequence determines all of i
ts higher levels of structure, much as the linear sequence of letters in books de
termines word, sentence, and paragraph structure. However, this analogy is an
oversimplification. Most proteins cannot fold properly by them selves outside
their normal cellular environment. Some cellular factors besides the protein it
self seem to be required in these cases, and folding often must occur during sy
nthesis of a polypeptide. What forces hold a protein in its proper shape? Some
of these are covalent bonds, but most are noncovalent. The principal covalent
bonds within and between polypeptides are disulfi de (S –S) bonds between c
ysteines. The noncovalent bonds are primarily hydrophobic and hydrogen bon
ds. Predictably, hydrophobic amino acids cluster together in the interior of a p
olypeptide, or at the interface between polypeptides, so they can avoid contact
with water ( hydrophobic, meaning water-fearing). Hydrophobic interactions
play a major role in tertiary and quaternary structures of proteins.
 Discovery of Messenger R
NA
• The concept of a messenger RNA carrying information from gene to
ribosome developed in stages during the years following the publicat
ion of Watson and Crick ’s DNA model. In 1958, Crick himself prop
osed that RNA serves as an intermediate carrier of genetic informati
on. He based his hypothesis in part on the fact that the DNA resides i
n the nucleus of eukaryotic cells, whereas proteins are made in the c
ytoplasm. This means that something must carry the information fro
m one place to the other. Crick noted that ribosomes contain RNA an
d suggested that this ribosomal RNA (rRNA) is the information bear
er. But rRNA is an integral part of ribosomes; it cannot escape. Ther
efore, Crick ’s hypothesis implied that each ribosome, with its own r
RNA, would produce the same kind of protein over and over.
• Fran çois Jacob and colleagues proposed an alternative hypothesis
calling for nonspecialized ribosomes that translate unstable RNAs
called messengers. The messengers are independent RNAs that bri
ng genetic information from the genes to the ribosomes. In 1961, J
acob, along with Brenner and Matthew Meselson, published their p
roof of the messenger hypothesis.
• This study used the same bacteriophage (T2) that Hershey and Cha
se had employed almost a decade earlier to show that genes were
made of DNA. The premise of the experiments was this: When pha
ge T2 infects E. coli, it subverts its host from making bacterial prot
eins to making phage proteins. If Crick ’s hypothesis were correct,
this switch to phage protein synthesis should be accompanied by th
e production of new ribosomes equipped with phage-specific RNA
s.
• To distinguish new ribosomes from old, these investigators labeled the
ribosomes in uninfected cells with heavy isotopes of nitrogen ( 15 N) a
nd carbon ( 13 C). This made “old ” ribosomes heavy. Then they infect
ed these cells with phage T2 and simultaneously transferred them to m
edium containing light nitrogen ( 14 N) and carbon ( 12 C). Any “new
” ribosomes made after phage infection would therefore be light and
would separate from the old, heavy ribosomes during density gradient
centrifugation. Brenner and colleagues also labeled the infected cells
with 32 P to tag any phage RNA as it was made. Then they asked this
question: Was the radio actively labeled phage RNA associated with ne
w or old ribosomes?
• Experimental test of the messenger hypothesis.
Heavy E. coli ribosomes were made by labeling
the bacterial cells with heavy isotopes of carbon
and nitrogen. The bacteria were then infected wi
th phage T2 and simultaneously shifted to “light
” medium containing the normal isotopes of car
bon and nitrogen, plus some 32 P to make the p
hage RNA radioactive. (a) Crick had proposed t
hat ribosomal RNA carried the message for mak
ing proteins. If this were so, then whole new rib
osomes with phage specific ribosomal RNA wo
uld have been made after phage infection. In tha
t case, the new 32 P-labeled RNA (green) shoul
d have moved together with the new, light ribos
omes (pink). (b) Jacob and colleagues had prop
osed that a messenger RNA carried genetic infor
mation to the ribosomes. According to this hypo
thesis, phage infection would cause the synthesi
s of new, phage-specific messenger RNAs that
would be 32 P-labeled (green). These would ass
ociate with old, heavy ribosomes (blue). The rad
ioactive label would therefore move together wi
th the old, heavy ribosomes in the density gradi
ent. This was indeed what happened.
• the phage RNA was found on old ribosomes whose rRNA was made before infectio
n even began. Clearly, this old rRNA could not carry phage genetic information; by
extension, it was very unlikely that it could carry host genetic information, either. T
hus, the ribosomes are constant. The nature of the polypeptides they make depends
on the mRNA that associates with them. This relationship resembles that of a DVD
player and DVD. The nature of the movie (polypeptide) depends on the DVD (mRN
A), not the player (ribosome).
• Other workers had already identifi ed a better candidate for the messenger: a class o
f unstable RNAs that associate transiently with ribosomes. Interestingly enough, in
phage T2-infected cells, this RNA had a base com position very similar to that of ph
age DNA —and quite different from that of bacterial DNA and RNA. This is exactl
y what we would expect of phage messenger RNA (mRNA) , and that is exactly wh
at it is. On the other hand, host mRNA, unlike host rRNA, has a base composition si
milar to that of host DNA. This lends further weight to the hypothesis that mRNA, n
ot rRNA, is the informational molecule.
 Transcription

• As you might expect, transcription follows the same base pairing rule
s as DNA replication: T, G, C, and A in the DNA pair with A, C, G, a
nd U, respectively, in the RNA product. (Notice that uracil appears in
RNA in place of thymine in DNA.) This base-pairing pattern ensures
that an RNA transcript is a faithful copy of the gene.
• Of course, highly directed chemical reactions such as transcription do
not happen at significant rates by themselves —they are enzyme-cata
lyzed. The enzyme that directs transcription is called RNA polymeras
e. Transcription has three phases: initiation, elongation, and terminati
on. The following is an outline of these three steps in bacteria:
• Making RNA. (a) Phosphodiester bond formation in RNA synthesis. ATP and GTP are joined
together to form a dinucleotide. Note that the phosphorus atom closest to the guanosine is ret
ained in the phosphodiester bond. The other two phosphates are removed as a by-product call
ed pyrophosphate. (b) Synthesis of RNA on a DNA template. The DNA template at top contai
ns the sequence 3’-dC-dA-dT-dG-5’ and extends in both directions, as indicated by the dashe
d lines. To start the RNA synthesis, GTP forms a base pair with the dC nucleotide in the DNA
template. Next, UTP provides a uridine nucleotide, which forms a base pair with the dA nucle
otide in the DNA template and forms a phosphodiester bond with the GTP. This produces the
dinucleotide GU. In the same way, a new nucleotide joins the growing RNA chain at each ste
p until transcription is complete. The pyrophosphate by-product is not shown.
• Transcription. (1a) In the first stage of initiation, RNA polymerase (red) binds tightly to the
promoter and “melts ” a short stretch of DNA. (1b) In the second stage of initiation, the pol
ymerase joins the first few nucleotides of the nascent RNA (blue) through phosphodiester b
onds. The first nucleotide retains its triphosphate group (ppp). (2) During elongation, the m
elted bubble of DNA moves with the polymerase, allowing the enzyme to “read ” the bases
of the DNA template strand and make complementary RNA. ( 3 ) Termination occurs when
the polymerase reaches a termination signal, causing the RNA and the polymerase to fall of
f the DNA template
• 1. Initiation First, the enzyme recognizes a region called a promoter,
which lies just “upstream ” of the gene. The polymerase binds tightly
to the promoter and causes localized melting, or separation, of the tw
o DNA strands within the promoter. At least 12 bp are melted. Next,
the polymerase starts building the RNA chain. The substrates, or buil
ding blocks, it uses for this job are the four ribonucleoside triphosph
ates: ATP, GTP, CTP, and UTP. The fi rst, or initiating,substrate is us
ually a purine nucleotide. After the first nucleotide is in place, the po
lymerase joins a second nucleotide to the fi rst, forming the initial ph
osphodiester bond in the RNA chain. Several nucleotides may be joi
ned before the polymerase leaves the promoter and elongation begins
.
• 2. Elongation During the elongation phase of transcription, RNA polymerase d
irects the sequential binding of ribonucleotides to the growing RNA chain in th
e 5’→3’ direction (from the 5’-end toward the 3’-end of the RNA). As it does s
o, it moves along the DNA template, and the “bubble ” of melted DNA moves
with it. This melted region exposes the bases of the template DNA one by one
so they can pair with the bases of the incoming ribonucleotides. As soon as the
transcription machinery passes, the two DNA strands wind around each other a
gain, re-forming the double helix. This points to two fundamental differences b
etween transcription and DNA replication: (a) RNA polymerase makes only on
e RNA strand during transcription, which means that it copies only one DNA st
rand in a given gene. (However, the opposite strand may be transcribed in anot
her gene.) Transcription is therefore said to be asymmetrical. This contrasts wit
h semiconservative DNA replication, in which both DNA strands are copied.
(b) In transcription, DNA melting is limited and transient. Only enough strand
separation occurs to allow the polymerase to “read ” the DNA template strand.
However, during replication, the two parental DNA strands separate permanent
ly.
• 3. Termination Just as promoters serve as initiation signals for transcr
iption, other regions at the ends of genes, called terminators, signal te
rmination. These work in conjunction with RNA polymerase to loose
n the association between RNA product and DNA template. The resul
t is that the RNA dissociates from the RNA polymerase and DNA, the
reby stopping transcription.

• A final, important note about conventions: RNA sequences are usuall

y written 5 9 to 3 9, left to right. This feels natural to a molecular biol
ogist because RNA is made in a 5’-to-3’ direction, and, as we will see
, mRNA is also translated 5’ to 3’. Thus, because ribosomes read the
message 5’ to 3’, it is appropriate to write it 5’ to 3’ so that we can rea
d it like a sentence.
• Genes are also usually written so that their transcription pro
ceeds in a left-to-right direction. This “flow ” of transcriptio
n from one end to the other gives rise to the term upstream,
which refers to the DNA close to the start of transcription
(near the left end when the gene is written conventionally).
Thus, we can describe most promoters as lying just upstrea
m of their respective genes. By the same convention, we sa
y that genes generally lie downstream of their promoters. G
enes are also conventionally written with their non template
strands on top.
 RNA Polymerase Structure

• As early as 1960–1961, RNA polymerases were discovere

d in animals, plants, and bacteria. And, as you might antici
pate, the bacterial enzyme was the fi rst to be studied in gr
eat detail. By 1969, the polypeptides that make up the E. c
oli RNA polymerase had been identifi ed by SDS polyacry
lamide gel electrophoresis (SDS-PAGE)
• Separation of s-factor from core E. coli RNA polymerase by phosphor cellulose chromatogra
phy. Burgess, Travers, and colleagues subjected RNA polymerase holoenzyme to phosphor c
ellulose chromatography, which yielded three peaks of protein: A, B, and C. Then they perfor
med SDS-PAGE on the holoenzyme (lane 1), peaks A, B, and C (lanes 2–4, respectively), and
purified σ (lane 5). Peak A contained σ, along with some contaminants (the most prominent o
f which is marked with an asterisk), B contained the holoenzyme, and C contained the functio
nal core polymerase (subunits α,β, and β’).
• lane 1, presents the results of an SDS-PAGE separation of the subunits of the E.
coli RNA polymerase by Richard Burgess, Andrew Travers, and their colleague
s. This enzyme preparation contained two very large subunits: beta (β) and beta-
prime (β’), with molecular masses of 150 and 160 kD, respectively. These two s
ubunits were not well separated in this experiment, but they were clearly disting
uished in subsequent studies. The other RNA polymerase subunits visible on thi
s gel are called sigma (σ) and alpha (α), with molecular masses of 70 and 40 kD
, respectively. Another subunit, omega (v), with a molecular mass of 10 kDa is n
ot detectable here, but was clearly visible in urea gel electrophoresis experiment
s performed on this same enzyme preparation. In contrast to the other subunits, t
he v-subunit is not required for cell viability, nor for enzyme activity in vitro. It
seems to play a role, though not a vital one, in enzyme assembly. The polypepti
de marked with an asterisk was a contaminant. Thus, the subunit content of an
RNA polymerase holoenzyme is β’, β, σ, σ2, ω; in other words, two molecules o
f a and one of all the others are present.
• lane 2 shows the composition of peak A, which contained t
he σ-subunit, along with a prominent contaminating polype
ptide and perhaps a bit of β’. Lane 3 shows the polypeptide
s in peak B, which contained the holoenzyme. Lane 4 show
s the composition of peak C, containing the core polymeras
e, which clearly lacks the σ-subunit. Further purification of
the s-subunit yielded the preparation in lane 5, which was f
ree of most contamination.
 Sigma (σ) as a Specificity Factor
• Adding s back to the core reconstituted the enzyme’s ability to transcr
ibe unnicked T4 DNA. Even more significantly, Ekkehard Bautz and c
olleagues showed that the holoenzyme transcribed only a certain class
of T4 genes (called immediate early genes), but the core showed no su
ch specificity.
• Not only is the core enzyme indiscriminate about the T4 genes it trans
cribes, it also transcribes both DNA strands. Bautz and colleagues de
monstrated this by hybridizing the labeled product of the holoenzyme
or the core enzyme to authentic T4 phage RNA and then checking for
RNase resistance.
• That is, they attempted to get the two RNAs to base-pair together an
d form an RNase-resistant double-stranded RNA. Because authentic
T4 RNA is made asymmetrically (only one DNA strand in any give
n region is copied), it should not hybridize to T4 RNA made properl
y in vitro because this RNA is also made asymmetrically and is ther
efore identical, not complementary, to the authentic RNA. Bautz an
d associates did indeed observe this behavior with RNA made in vit
ro by the holoenzyme. However, if the RNA is made symmetrically
in vitro, up to half of it will be complementary to the in vivo RNA a
nd will be able to hybridize to it and thereby become resistant to R
Nase. In fact, Bautz and associates found that about 30% of the labe
led RNA made by the core polymerase in vitro became RNase-resist
ant after hybridization to authentic T4 RNA. Thus, the core enzyme
acts in an unnatural way by transcribing both DNA strands
• Next, the investigators tested the RNA polymerase activities of the
two separated components of the enzyme: the core polymerase an
d the σ-factor. The above table shows that this separation had caus
ed a profound change in the enzyme’s activity. Whereas the holoen
zyme could transcribe intact phage T4 DNA in vitro quite actively,
the core enzyme had little ability to do this. On the other hand, cor
e polymerase retained its basic RNA polymerizing function becaus
e it could still transcribe highly nicked templates (DNAs with sing
le-stranded breaks) very well. (As we will see, transcription of nic
ked DNA is a laboratory artifact and has no biological significanc
e.)
• Clearly, depriving the holoenzyme of its σ-subunit leaves a
core enzyme with basic RNA synthesizing capability, but l
acking specificity. Adding s back restores specificity. In fa
ct, σ was named only after this characteristic came to light,
and the σ, or Greek letter s, was chosen to stand for “specif
icity”
 Promoters

• The polymerase binding sites are called promoters

Binding of RNA Polymeras
e to Promoters

• RNA polymerase/promoter binding. (a) The holoenzyme binds and rebinds loosely t
o the DNA, searching for a promoter. (b) The holoenzyme has found a promoter and
has bound loosely, forming a closed promoter complex. (c) The holoenzyme has bo
und tightly, melting a local region of DNA and forming an open promoter complex.
• RNA polymerase holoenzyme binds loosely to DNA at
first. It either binds initially at a promoter or scans alon
g the DNA until it finds one. The complex with holoenz
yme loosely bound at the promoter is called a closed pr
omoter complex because the DNA remains in closed do
uble-stranded form. Then the holoenzyme can melt a sh
ort region of the DNA at the promoter to form an open
promoter complex in which the polymerase is bound ti
ghtly to the DNA. This is called an open promoter com
plex because the DNA has to open up to form it.
• It is this conversion of a loosely bound polymerase in
a closed promoter complex to the tightly bound polym
erase in the open promoter complex that requires s, an
d this is also what allows transcription to begin. We ca
n now appreciate how σ fulfills its role in determining
specificity of transcription: It selects the promoters to
which RNA polymerase will bind tightly. The genes ad
jacent to these promoters will then be transcribed.
 Promoter Structure
• What is the special nature of a bacterial promoter that attra
cts RNA polymerase? David Pribnow compared several E.
coli and phage promoters and discerned a region they held
in common: a sequence of 6 or 7 bp centered approximatel
y 10 bp upstream of the start of transcription. This was ori
ginally dubbed the “Pribnow box,” but is now usually calle
d the -10 box. Mark Ptashne and colleagues noticed anothe
r short sequence centered approximately 35 bp upstream of
the transcription start site; it is known as the -35 box. Thou
sands of promoters have now been examined and a typical,
or consensus sequence for each of these boxes has emerge
d
• A bacterial promoter. The positions of -10 and -35 boxes a
nd the unwound region are shown relative to the start of tra
nscription for a typical E. coli promoter. Capital letters den
ote bases found in those positions in more than 50% of pro
moters examined; lower-case letters denote bases found in
those positions in 50% or fewer of promoters examined.
• These so-called consensus sequences represent probabilities. The capi
tal letters denote bases that have a high probability of being found in t
he given position. The lowercase letters correspond to bases that are u
sually found in the given position, but at a lower frequency than those
denoted by capital letters. The probabilities are such that one rarely fi
nds -10 or -35 boxes that match the consensus sequences perfectly. H
owever, when such perfect matches are found, they tend to occur in v
ery strong promoters that initiate transcription unusually actively. In
fact, mutations that destroy matches with the consensus sequences te
nd to be down mutations. That is, they make the promoter weaker, res
ulting in less transcription. Mutations that make the promoter sequen
ces more like the consensus sequences usually make the promoters st
ronger; these are called up mutations. The spacing between promoter
elements is also important, and deletions or insertions that move the -
10 and -35 boxes unnaturally close together or far apart are deleteriou
s.
• In addition to the -10 and -35 boxes, which we can call cor
e promoter elements, some very strong promoters have an
additional element farther upstream called an UP element.
E. coli cells have seven genes (rrn genes) that encode rRN
As. Under rapid growth conditions, when rRNAs are requi
red in abundance, these seven genes by themselves accoun
t for the majority of the transcription occurring in the cell.
Obviously, the promoters driving these genes are extraordi
narily powerful, and their UP elements are part of the expl
anation.
• The rrnB P1 promoter. The core promoter elements (210 a
nd 235 boxes, blue) and the UP element (red) are shown sc
hematically above, and with their complete base sequences
(nontemplate strand) below, with the same color coding.
• shows the structure of one of these promoters, the rrnB P1
promoter. Upstream of the core promoter (blue), there is an
UP element (red) between positions -40 and -60. We know
that the UP element is a true promoter element because it s
timulates transcription of the rrnB P1 gene by a factor of 3
0 in the presence of RNA polymerase alone. Because it is r
ecognized by the polymerase itself, we conclude that it is a
promoter element. This promoter is also associated with th
ree so-called Fis sites between positions -60 and -150, whi
ch are binding sites for the transcription-activator protein F
is. The Fis sites, because they do not bind to RNA polymer
ase itself, are not classical promoter elements, but instead a
re members of another class of transcription- activating D
NA elements called enhancers.
Transcription Initiation

• Stages of transcription initiatio

n. (a) RNA polymerase binds t
o DNA in a closed promoter c
omplex. (b) The s-factor stimu
lates the polymerase to conver
t the closed promoter complex
to an open promoter complex.
(c) The polymerase incorporat
es the first 9 or 10 nt into the n
ascent RNA. Some abortive tr
anscripts are pictured at left.
(d) The polymerase clears the
promoter and begins the elong
ation phase. The σ-factor may
be lost at this point or later, du
ring elongation.
• (1) formation of a closed promoter complex; (2) conversio
n of the closed promoter complex to an open promoter co
mplex; (3) polymerizing the first few nucleotides (up to 1
0) while the polymerase remains at the promoter, in an initi
al transcribing complex; and (4) promoter clearance, in wh
ich the transcript becomes long enough to form a stable hy
brid with the template strand. This helps to stabilize the tra
nscription complex, and the polymerase changes to its elon
gation conformation and moves away from the promoter. I
n this section, we will examine the initiation process in mo
re detail.
Local DNA Melting at the
Promoter
• Locating the region of a T7 phage early promoter melted by RNA polymera
se. (a) When adenine is base-paired with thymine (left) the N1 nitrogen of a
denine is hidden in the middle of the double helix and is therefore protected
from methylation. On melting (right), the adenine and thymine separate; thi
s opens the adenine up to attack by dimethyl sulfate (DMS, blue), and the N
1 nitrogen is methylated. Once this occurs, the methyl-adenine can no longe
r base-pair with its thymine partner. (b) A hypothetical promoter region con
taining five A–T base pairs is end-labeled (orange), then RNA polymerase
(red) is bound, which causes local melting of the promoter DNA. The three
newly exposed adenines are methylated with dimethyl sulfate (DMS). Then
, when the polymerase is removed, the A–T base pairs cannot reform becau
se of the interfering methyl groups (m, blue). Now S1 nuclease can cut the
DNA at each of the unformed base pairs because these are local single-stran
ded regions. Very mild cutting conditions are used so that only about one cu
t per molecule occurs. Otherwise, only the shortest product would be seen.
The resulting fragments are denatured and electrophoresed to determine the
ir sizes. These sizes tell how far the melted DNA region was from the labele
d DNA end.
• On binding to a promoter, RNA polymerase causes melting
that has been estimated at 10–17 bp in the vicinity of the tr
anscription start site. This transcription bubble moves with
the polymerase, exposing the template strand so it can be tr
anscribed.
 Elongation

• After initiation of transcription is accomplished, the core continues to

elongate the RNA, adding one nucleotide after another to the growing
RNA chain. In this section we will explore this elongation process.

• In this section we will see evidence that the β- and β’ -subunits are in
volved in phosphodiester bond formation, that these subunits also part
icipate in DNA binding, and that the a-subunit has several activities, i
ncluding assembly of the core polymerase.
• Separation and reconstitution of RNA polymerase to locate the determinant of antibi
otic resistance. Start with RNA polymerases from rifampicin-sensitive and -resistant
E. coli cells, separate them into their component polypeptides, and recombine them i
n various combinations to reconstitute the active enzyme. In this case, the α-, β’-, an
d σ-subunits came from the rifampicin-sensitive polymerase (blue), and the b-subuni
t came from the antibiotic-resistant enzyme (red). The reconstituted polymerase is rif
ampicin-resistant, which shows that the b-subunit determines sensitivity or resistanc
e to this antibiotic
 The Role of β in Phosphodiester Bond
Formation
• Walter Zillig was the first to investigate the individual core subunits, in 1970
. He began by separating the E. coli core polymerase into its three componen
t polypeptides and then combining them again to reconstitute an active enzy
me. The separation procedure worked as follows: Alfred Heil and Zillig elect
rophoresed the core enzyme on cellulose acetate in the presence of urea. Lik
e SDS, urea is a denaturing agent that can separate the individual polypeptid
es in a complex protein. Unlike SDS, however, urea is a mild denaturant that
is relatively easy to remove. Thus, it is easier to renature a urea-denatured po
lypeptide than an SDS denatured one. After electrophoresis was complete, H
eil and Zillig cut out the strips of cellulose acetate containing the polymerase
subunits and spun them in a centrifuge to drive the buffer, along with the pro
tein, out of the cellulose acetate. This gave them all three separated polypept
ides, which they electrophoresed individually to demonstrate their purity
• Purification of the individual subunits of E. coli RNA poly
merase. Heil and Zillig subjected the E. coli core polymera
se to urea gel electrophoresis on cellulose acetate, then coll
ected the separated polypeptides. Lane 1, core polymerase
after electrophoresis; lane 2, purified α; lane 3, purified β; l
ane 4, purified β’.
Termination
of Transcription

• When the polymerase reaches a terminator at the end of a

gene it falls off the template, releasing the RNA. E. coli cel
ls contain about equal numbers of two kinds of terminators
. The first kind, known as intrinsic terminators, function wi
th the RNA polymerase by itself without help from other p
roteins. The second kind depend on an auxiliary factor call
ed rho . Naturally, these are called rho dependent terminato
rs. Let us consider the mechanisms of termination employe
d by these two systems, beginning with the simpler, intrins
ic terminators.
Rho-Independent Ter
mination
• Rho-independent, or intrinsic, termination depends on term
inators consisting of two elements: an inverted repeat follo
wed immediately by a T-rich region in the non template str
and of the gene. The model of termination we will present
later in this section depends on a “hairpin” structure in the
RNA transcript of the inverted repeat. Before we get to the
model, we should understand how an inverted repeat predi
sposes a transcript to form a hairpin.
• Inverted Repeats and Hairpins Consider this inverted repea
t:

• Such a sequence is symmetrical around its center, indicate

d by the dot; it would read the same if rotated 180 degrees
in the plane of the paper, and if we always read the strand t
hat runs 5’→3’ left to right. Now observe that a transcript
of this sequence
• is self-complementary around its center (the underlined G).
That means that the self-complementary bases can pair to f
orm a hairpin as follows:

The A and the U at the apex of the hairpin cannot form

a base pair because of the physical constraints of the turn
in the RNA.
• The Structure of an Intrinsic Terminator
• The E. coli trp operon contains a DNA sequence called an attenuat
or that causes premature termination of transcription. The trp atten
uator contains the two elements (an inverted repeat and a string of
T’s in the nontemplate DNA strand) suspected to be vital parts of a
n intrinsic terminator, so Peggy Farnham and Terry Platt used atten
uation as an experimental model for normal termination. The inver
ted repeat in the trp attenuator is not perfect, but 8 bp are still possi
ble, and 7 of these are strong G–C pairs, held together by three hyd
rogen bonds. The hairpin looks like this:
A Model for Termination
• A model for rho-independent, or intrinsi
c termination. (a) The polymerase has p
aused at a string of weak rU–dA base pa
irs, and a hairpin has started to form just
upstream of these base pairs. (b) As the
hairpin forms, it further destabilizes the
RNA–DNA hybrid. This destabilization
could take several forms: The formation
of the hairpin could physically pull the
RNA out of the polymerase, allowing th
e transcription bubble to collapse; conve
rsely, it could cause the transcription bu
bble to collapse, expelling the RNA fro
m the hybrid. (c) The RNA product and
polymerase dissociate completely from t
he DNA template, terminating transcript
ion.
Rho-Dependent Term
ination
• A model of rho-dependent terminatio
n. (a) Rho (blue) has joined the elong
ation complex by binding directly to
RNA polymerase. The end of the nasc
ent transcript (green) has just emerge
d from the polymerase. (b) The transc
ript has lengthened and has bound to
rho via a rho loading site, forming an
RNA loop. Rho can now feed the tran
script through its central cavity. (c) T
he polymerase has paused at a termin
ator. By continuously feeding the tran
script through itself, rho has tightened
the RNA loop and irreversibly trappe
d the elongation complex. Rho has al
so begun to dissociate the RNA–DN
A hybrid, which will lead to transcrip
t release.
 SUGGESTED READINGS

• Busby, S. and R.H. Ebright. 1994. Promoter structure, pro

moter recognition, and transcription activation in prokaryo
tes. Cell 79:743–46.

• Roberts, J.W. 2006. RNA polymerase, a scrunching machi

ne. Science 314:1097–98
 Self study

• The lac Operon Negative control of the lac operon. (a) No lactose; repression. The l
acI gene produces repressor (green), which binds to the operator and blocks RNA p
olymerase from transcribing the lac genes. (b) Presence of lactose, derepression. Th
e inducer (black) binds to repressor, changing it to a form (bottom) that no longer bi
nds well to the operator. This removes the repressor from the operator, allowing RN
A polymerase to transcribe the structural genes. This produces a polycistronic mRN
A that is translated to yield β-galactosidase, permease, and transacetylase