Motiffs

Gene
mRNA
Translation at ribosome
Protein
Transcription
Transcription: DNA -> RNA

DNA RNA
Protein
Transcription
Translation
1)tRNA
2)rRNA
3)snRNA
4)mRNA
mRNA is the only type of
RNA that is translated into
protein

©1998 by Alberts, Bray, Johnson, Lewis, Raff, Roberts, Walter.
Published by Garland Publishing, a member of the Taylor & Francis Group.

The Population of mRNA Molecules in a Typical
Mammalian Cell
----------------------------------------------------------------------------------------------------------
Copies/cell#/class Total mRNA molecules/class
Abundant 12,000 4 = 48,000
Intermediate 300 500 = 150,000
Scarce 15 11,000 = 165,000
----------------------------------------------------------------------------------------------------------
This division of mRNAs into just three discrete classes is somewhat arbitrary, and in
many cells a more continuous spread in abundances is seen. However, a total of 10,000
to 20,000 different mRNA species is normally observed in each cell, most species being
present at a low level (5 to 15 molecules per cell). Most of the total cytoplasmic RNA is
rRNA, and only 3% to 5% is mRNA, a ratio consistent with the presence of about 10
ribosomes per mRNA molecule. This particular cell type contains a total of about
360,000 mRNA molecules in its cytoplasm.
----------------------------------------------------------------------------------------------------------

The key steps in transcription
– Initiation
– Elongation
– Termination
DNA
+
RNA

Reaction mechanism of RNA polymerase
5. RNA polymerases don’t need a primer, but do need ds DNA
1. DNA-dependent RNA Polymerases
2. RNA Pols polymerize in the 5’ to 3’ direction (rNTP added only to the 3’ end)
3. 3’ OH of chain reacts with the a PO4 of incoming rNTP, liberating pyrophosphate
4. Added ribonucleotide follows Watson-Crick pairing rules, determined by template strand
6. RNA polymerase lacks exonuclease activities, then can not proof-read and is much more
error prone than DNA polymerase.

Schematic representation of the subunit structure of yeast nuclear RNA polymerases
and comparison with E. coli RNA core polymerase.

Subunit structure of purified nuclear
RNA polymerases (nRNAP)
• All 3 have 10-14 subunits.
• Subunits range from 10 to 220 kDa.
• All 3 have 2 very large (>125 kD) subunits
and several smaller ones.
• Several of the smaller subunits (5 in
yeast) are common to all 3 Pol.

Promoter
RNA
5’ 3’
Where is transcription
initiated?
• Promoters are sequences in the DNA just
upstream of transcripts (coding sequences) that
define the sites of initiation
• The role of the promoter is to attract RNA
polymerase to the correct start site so
transcription can be initiated

S1 mapping of the 5’ end of a RNA Transcript
A 5’ end labeled single-stranded DNA probe is prepared from the
template strand. After hybridization to RNA and digestion with S1,
the size of the protected probe tells approx. where transcription
started.

High resolution analysis of
the 5’end of an RNA
transcript by primer
extension.
Primer is an end-labeled DNA
oligonucleotide (~20 nt) that is
complementary to a sequence
in the RNA ~150 nt from the
expected 5’ end.
Lane E- extended DNA product
Lanes A,C, G, T – sequence
ladder generated with the same
oligo primer, but on the
corresponding cloned DNA.

Mapping DNA-Protein interactions

Biochemical approaches to
defining promoter sites
• DNAse footprinting can be used to identify sites
where RNA polymerase is in close contact with DNA.
How we can generate this end labeled DNA?

Sample of a DNAse I
footprinting gel (for a
DNA-binding protein).
Footprint
Lanes 2-4 had
increasing amounts of
the DNA-binding
protein (lambda
protein cII); lane 1 had
none.

Dimethylsulfate (DMS) Footprinting
1. End-label DNA fragment.
2. Bind protein.
3. Treat with dimethylsulfate,
which methylates purine
bases.
4. Partially cleave DNA by
depurinating the methylated
bases (piperidine)
5. Separate DNA fragments on
DNA sequencing gels.

Sample of DMS footprinting.
Lanes 1 and 4 had no protein
Lanes 2 and 3 had 2 different
amounts of protein.
Protein binding protects some purines
from modification by DMS, it but can
stimulate modification of others (helix
distorted or partially melted).

An electrophorectic mobility shift assay (EMSA) The principle of the assay is shown
schematically in (A). In this example an
extract of an antibody-producing cell line
is mixed with a radioactive DNA fragment
containing about 160 nucleotides of a
regulatory DNA sequence from a gene
encoding the light chain of the antibody
made by the cell line. The effect of the
proteins in the extract on the mobility of
the DNA fragment is analyzed by
polyacrylamide-gel electrophoresis
followed by autoradiography. The free
DNA fragments run rapidly to the bottom
of the gel, while those fragments bound to
proteins are retarded; the finding of six
retarded bands suggests that the extract
contains six different sequence-specific
DNA-binding proteins (indicated as C1-
C6) that bind to this DNA sequence. (For
simplicity, any DNA fragments with more
than one protein bound have been omitted
from the figure.) In (B) the extract was
fractionated by a standard chromato-
graphic technique (top), and each fraction
was mixed with the radio-active DNA
fragment, applied to one lane of a
polyacrylamide gel, and analyzed as in
(A). (B, modified from C. Scheidereit, A.
Heguy, and R.G. Roeder, Cell 51:783-793,
1987. © Cell Press.)
Navigation

DNA affinity chromatography
HOW WE CAN PURIFY A DNA BINDING PROTEIN?

Promoter
RNA
5’ 3’
Bioinformatics approaches to
defining promoter sites
• Comparison of known start sites to identify
consensus sequences:

Promoter
RNA
5’ 3’
• Regions of similarity are found around 10 and 35 bases before the start site of
transcription:
• DNAse protection shows that RNA polymerase can bind to these same regions.
• Mutations of these sites can lead to the elimination or reduction of transcriptional
initiation at a promoter.
• Differences in these sites control the relative rates of expression of different genes.
• Strong promoters have sites that are very similar to the consensus sequence while
weak promoters show many differences

Schematic diagram of the steps in the
initiation of RNA synthesis (DNA
transcription) catalyzed by RNA
polymerase.
The enzyme first forms a closed complex in
which the two DNA strands remain fully base-
paired. In the next step the enzyme catalyzes the
opening of a little more than one turn of the
DNA helix to form an open complex, in which
the template DNA strand is exposed for the
initiation of an RNA chain. The polymerase
containing the bound σ subunit, however,
behaves as though it is tethered to the promoter
site: it seems unable to proceed with the
elongation of the RNA chain and on its own
frequently synthesizes and releases short RNA
chains. As indicated, the conversion to an
actively elongating polymerase requires the
release of initiation factors (the sigma subunit in
the case of the E. coli enzyme) and generally
involves the binding of other proteins that serve
as elongation factors.
How does sigma associate with a promoter?
The σ subunit appears to have two segments that contact the bases of DNA molecule
through the major groove. It does this while it is associated with the core enzyme.
-10-35
How does σ70
promote binding of RNA polymerase to
promoters?
• σ70
lowers the general affinity of RNA
polymerase for DNA.
– As a result, RNA polymerase is able
to move quickly along DNA
scanning for promoter sites.
• σ70
can bind specifically to promoters (the
-10 and -35 regions).
– This allows the holoenzyme to bind
tightly to promoters when they are
encountered.
• RNA polymerase searches for promoter
sites by moving along the DNA rather
than by searching randomly throughout
the cell.

The elongation stage
• σ70
dissociates from the core RNA polymerase after
initiation occurs. This yields:
• In the absence of σ70
, RNA polymerase binds ssDNA tightly
and is highly processive.

4. Termination
Two types of termination events in E. coli
– Rho independent
– Rho dependent

IR in DNA produces a stem-loop in RNA.

Stem-loop
formation
competes with
the RNA-DNA
hybrid (Open
Complex).
Causing DNA
helix to reform
(Closed
complex).

Rho-dependent termination
• Some mRNAs synthesized by RNA polymerase in vitro fail to
terminate at the normal in vivo position.
– This suggested that additional proteins might be required for
termination at these sites.
– The missing factor was identified and named rho.

Rho in action
Rho binds
transcripts at
stretches of ~100
nt free of 2nd
structure and rich
in cytosines.
Rho is a hexamer
helicase.
Can unwind RNA-DNA
hybrids.

Studies of RNA synthesis by
isolated nuclei
• RNA synthesis by isolated nuclei indicated that
there were at least 2 polymerases; one of which
was in the nucleolus and synthesized rRNA
– rRNA often has a higher G-C content than other
RNAs; a G-C rich RNA fraction was preferentially
synthesized with low ionic strength and Mg2+
– Another less G-C rich RNA fraction was preferentially
synthesized at higher ionic strength with Mn2+

A protein extract from the nuclei of
cultured frog cells was passed through
a DEAE Sephadex column to which
charged proteins absorb differentially.
Adsorbed proteins were eluted (black
curve) with a solution of constantly
increasing NaCl concentration.
Fractions containing the eluted proteins
were assayed for the ability to
transcribe DNA (red curve) in the
presence of the four ribonucleoside
triphosphates. The synthesis of RNA by
each fraction in the presence of 1 ug/ml
of α-amanitin also was measured (blue
curve).
[See R. G. Roeder, 1974, J. Biol.
Chem. 249:241.]
Separation and identification of the three eukaryotic RNA polymerases by
column chromatography.

Determining roles for each
polymerase
• Purified polymerases don’t transcribe DNA
specifically – so used nuclear fractions.
• Also useful were two transcription inhibitors
1. α-aminitin – from a mushroom, inhibits Pol
II, and Pol III at higher concentrations.
2. Actinomycin D - general transcription
inhibitor, binds DNA and intercalates into
helix, prefers G-C rich regions (like rRNA
genes).

Drugs that inhibit RNA polymerases
α-amanitin: actinomycin D:

α-amanitin:
Pol II: K0.5 = 0.02 ug/ml
Pol III: K0.5 = 0.20 ug/ml
Po1 I: insensitive
actinomycin D:
Pol I most sensitive, but all three Pol's inhibited at
higher concentrations
Drug sensitivities
RNA Polymerase I:
1. Not inhibited by aminitin,
but inhibited by low
concentrations of
actinomycin D.
2. RNA produced in the
presence of α-
aminitin could be
competed by rRNA
for hybridization to
(rat) DNA.
Conclusion: Pol I
synthesizes the rRNA
precursor (45S pre-
rRNA  28S + 18S +
5.8S rRNAs)
RNA Polymerase II
1. Actinomycin D, at low
concentrations, did
not inhibit synthesis
of heterogenous
nuclear RNA (hn
RNA).
2. α-aminitin inhibited
synthesis of hnRNA
in nucleoplasmic
fraction.
Conclusion: Pol II
synthesizes hnRNA
(mostly mRNA
precursors).
RNA Polymerase III
Synthesis of small
abundant RNAs
inhibited only at high
[α-aminitin] Small
RNAs: tRNA
precursors, 5S rRNA,
U6 (involved in
splicing), and 7SL
RNA (involved in
protein secretion
through the ER, part
of the signal
recognition particle).
Conclusion: Pol III
synthesizes many of
the small abundant
cytoplasmic and
nuclear RNAs

HOW WE CAN MEASURE
TRANSCRIPTIONAL ACTIVITY IN VIVO?

Nascent-chain (run-on) assay for transcription rate of a gene. Isolated nuclei are
incubated with 32P-labeled ribonucleoside triphosphates for a brief period. During this period
RNA polymerase molecules that were transcribing a gene when the nuclei were isolated add
300 – 500 nucleotides to nascent RNA chains. Very little new initiation occurs. By
hybridizing the labeled RNA to the cloned DNA for a specific gene (A in this case), the
fraction of total RNA produced from that gene (i.e., its relative transcription rate) can be
measured. [See J. Weber et al., 1977, Cell 10:611.]

In vivo assay for transcription factor activity.
The assay system requires two plasmids.
One plasmid contains the gene encoding
the putative transcription factor (X
protein). The second plasmid contains a
reporter gene and one or more binding
sites for X protein. Both plasmids are
simultaneously introduced into host cells
that lack the gene encoding X protein and
the reporter gene. The production of
reporter-gene RNA transcripts is
measured; alternatively, the activity of
the encoded protein can be assayed. If
reporter-gene transcription is greater in
the presence of the X-encoding plasmid,
then the protein is an activator; if
transcription is less, then it is a repressor.
By use of plasmids encoding a mutated
or rearranged transcription factor,
important domains of the protein can be
identified. Cis-acting DNA sequences
can be identified by mutational analysis.

Use of linker scanning mutations to identify
transcription-control elements

General pattern of cis-acting control elements that
regulate gene expression in yeast and metazoans
(a) Genes of multicellular organisms contain both promoter-proximal elements and enhancers as
well as a TATA box or other promoter element. The latter positions RNA polymerase II to
initiate transcription at the start site and influences the rate of transcription. Enhancers may be
either upstream or downstream and as far away as 50 kb from the transcription start site. In some
cases, promoter-proximal elements occur downstream from the start site as well. (b) Most yeast
genes contain only one regulatory region, called an upstream activating sequence (UAS), and a
TATA box, which is ≈90 base pairs upstream from the start site.

Pol II basic promoter elements
- 10 0 -5 0 +1 5 0 100
core promote r
CAGAGC ATATAAGGTGAGGTAGG ATCAGTTGCTC CTCAC CTT
-30 -20 -10 + 1
TA TA -box Inr
Defines where transcription starts.
Also required for efficient transcription for some promoters.
Some class II promoters don’t have a TATA box.

Transcription starts at a purine ~25-30 bp from the TATA box.Transcription starts at a purine ~25-30 bp from the TATA box.
SV40 early
promoter
analyzed
in vivo.
Normal promoter.
The arrows indicate transcription start sites as determined by S1 mapping and primer extension.

TATA box also important for transcription
efficiency for some promoters.
Rabbit globin promoter, tested in Hela cells,
and assayed by S1 mapping of transcript 5’
end.

How the different base pairs in DNA can be recognized from
their edges without the need to open the double helix
The four possible
configurations of base
pairs are shown, with
hydrogen bond donors
indicated in blue,
hydrogen bond acceptors
in red, and hydrogen
bonds themselves as a
series of short parallel
redlines. Methyl groups,
which form hydrophobic
protuberances, are shown
in yellow, and hydrogen
atoms that are attached to
carbons, and are
therefore unavailable for
hydrogen bonding, are
white.
Figure and text modified
from Alberts et al.,
Molecular Biology of the
Cell (1994).

A DNA recognition code. The edge of each
base pair, seen here looking directly at the major
or minor groove, contains a distinctive pattern of
hydrogen bond donors, hydrogen bond
acceptors, and methyl groups. From the major
groove, each of the four base-pair configurations
projects a unique pattern of features. From the
minor groove, however, the patterns are similar
for G-C and C-G as well as for A-T and T-A.
The binding of a gene regulatory protein to
the major groove of DNA. Only a single type of
contact is shown. Typically, the protein-DNA
interface would consist of 10 to 20 such
contacts, involving different amino acids, each
contributing to the binding energy of the protein-
DNA interaction. Figure and text modified from Alberts et
al., Molecular Biology of the Cell (1994).

The DNA-binding helix-turn-helix motif.
The motif is shown in (A), where each white circle denotes the central carbon of an amino acid. The carboxyl-
terminal α-helix(red) is called the recognition helix because it participates in sequence-specific recognition of
DNA. As shown in (B), this helix fits into the major groove of DNA, where it contacts the edges of the base
pairs.
Figure and text modified from Alberts et al., Molecular Biology of the Cell (1994).

Some helix-turn-helix DNA-binding proteins
All of the proteins bind DNA as dimers in which the two copies of the recognition helix (red cylinder)
are separated by exactly one turn of the DNA helix (3.4 nm). The second helix of the helix-turn-helix
motif is colored blue. The lambda repressor and cro proteins control bacteriophage lambda gene
expression, and the tryptophan repressor and the catabolite activator protein (CAP) control the
expression of sets of E. coli genes. Figure and text modified from Alberts et al., Molecular Biology of the Cell
(1994).

Zinc Finger Protein
This protein belongs to the Cys-Cys-His-His family of zinc finger proteins, named after the amino acids
that grasp the zinc. This zinc finger is from a frog protein of unknown function. (A) Schematic drawing of
the amino acid sequence of the zinc finger. (B) The three-dimensional structure of the zinc finger is
constructed from an antiparallel β-sheet (amino acids 1 to 10) followed by an α-helix (amino acids 12 to
24). The four amino acids that bind the zinc (Cys 3, Cys 6, His 19, and His 23) hold one end of the α-
helix firmly to one end of the β-sheet. (Adapted from M.S. Lee et al., Science 245:635-637, 1989. © 1989
the AAAS.) Figure and text modified from Alberts et al., Molecular Biology of the Cell (1994).

DNA binding by a zinc finger protein
(A) The structure of a fragment of a mouse gene regulatory protein bound to a specific DNA site. This
protein recognizes DNA using three zinc fingers of the Cys-Cys-His-His type arranged as direct
repeats. (B) The three fingers have similar amino acid sequences and contact the DNA in similar ways.
In both (A) and (B) the zinc atom in each finger is represented by a small sphere. (Adapted from N.
Pavletich and C. Pabo, Science252:810-817, 1991. © 1991 the AAAS.). Figure and text modified from
Alberts et al., Molecular Biology of the Cell (1994).

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