Liam.Keegan@ceitec.muni.cz
Lewin, GENES XI
Chapter 2
Genes Encode RNAs and Polypeptides
Lewin’s GENES XII (PDF)
https://pdfroom.com/books/lewins-genes-xii/Wx5aDYKl2BJ
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Figure 02.01: Each chromosome has a single long molecule of DNA within which are the
sequences of individual genes.
‘Compare and contrast’
transcription in prokaryotes and eukaryotes
2.2 Most Genes Encode Polypeptides
• Ribosomal RNAs, tRNAs and ribosomal proteins are the
most highly-expressed gene products
• The one gene : one enzyme hypothesis summarizes
the basis of modern genetics: that a gene is a stretch of
DNA encoding one or more isoforms of a single
polypeptide chain. (Beadle and Tatum)
• one gene : one polypeptide hypothesis – A modified
version of the not generally correct one gene : one
enzyme hypothesis; the hypothesis that a gene is
responsible for the production of a single polypeptide.
(Vernon Ingram, 1957)
2.10 Bacterial Genes Are Colinear with
Their Proteins
• A bacterial gene
consists of a continuous
length of 3N nucleotides
that encodes N amino
acids.
• The gene is colinear
with both its mRNA and
polypeptide product,
• i.e. no introns Figure 02.12: The recombination map of
the tryptophan synthetase gene
corresponds with the amino acid
sequence of the polypeptide.
2.11 Transcription and translation are Required to
Express the Product of a Gene
• Each mRNA consists of a untranslated 5′ region (5′ UTR
or leader), a coding region, and an untranslated 3′ UTR
or trailer. Eukaryotes have introns and pre-mRNA
process ing to make longer-lasting mRNAs.
Figure 02.14: The gene is usually longer than the sequence encoding the polypeptide.
26.1 Introduction
• coupled transcription/translation – The phenomena in
bacteria where translation of the mRNA occurs
simultaneously with its transcription (40 nucleotides /sec,
2.4 kb / minute). In eukaryotes transcription and
translation are separated to nucleus and cytoplasm.
• Bacterial mRNA has the bare 5’ triphosphate from
transcription-initiation and ribonucleases ,5’-> 3’,
exonucleases, rapidly degrade mRNA behind
translating ribosomes
26.2 Operons, in prokaryotes only, are Structural Gene
Clusters that Are Coordinately Controlled
• Genes coding for proteins that function in the same
pathway may be located adjacent to one another and
controlled as a single unit that is transcribed into a
polycistronic mRNA.
Figure 26.05: The lac operon occupies ~6000 bp of DNA.
Lewin XI
Chapter 19
Prokaryotic Transcription
19.1 Introduction
• Transcription is 5′ to 3′ on a template that is 3′ to 5′.
• coding (nontemplate) strand – The DNA strand that
has the same sequence as the mRNA and is related by
the genetic code to the protein sequence that it
represents.
• RNA polymerase – An enzyme that synthesizes RNA
using a DNA template (formally described as a DNAdependent
RNA polymerase).
Figure 19.01: The function of RNA
polymerase is to copy one strand of
duplex DNA into RNA.
19.1 Introduction
• promoter – A region of DNA
where RNA polymerase binds to
initiate transcription.
• Start point – The position on
DNA corresponding to the first
base incorporated into RNA.
• terminator – A sequence of DNA
that causes RNA polymerase to
terminate transcription.
• transcription unit – The
sequence between sites of
initiation and termination by RNA
polymerase; it may include more
than one gene.
Figure 19.02: A transcription unit is a
sequence of DNA transcribed into a
single RNA, starting at the promoter
and ending at the terminator.
19.2 Transcription Occurs by Base Pairing
in a “Bubble” of Unpaired DNA
Figure 19.03: DNA strands separate to
form a transcription bubble. RNA is
synthesized by complementary base
pairing with one of the DNA strands.
Figure 19.05: During transcription, the
bubble is maintained within bacterial
RNA polymerase.
19.3 The Transcription Reaction Has
Three Stages
• RNA polymerase initiates transcription (initiation) after
opening the DNA duplex to form a transcription bubble
(the open complex).
• During elongation the transcription bubble moves along
DNA and the RNA chain is extended in the 5′→3′
direction by adding nucleotides to the 3′ end.
• Transcription stops (termination) and the DNA duplex
reforms when RNA polymerase dissociates at a
terminator site.
19.3 The Transcription Reaction Has
Three Stages
• Transcription initiation
• RNA polymerase binds to a
promoter site on DNA to form
a closed complex.
• RNA polymerase initiates
transcription (initiation) after
opening the DNA duplex to
form a transcription bubble
(the open complex).
Figure 19.06: Transcription has three stages.
19.17 Negative Supercoiling facilitates Transcription
and transcription affects supercoiling
• Negative supercoiling increases the efficiency of some
promoters by assisting the melting reaction.
• Transcription generates positive supercoils ahead of the
enzyme and negative supercoils behind it, and these
must be removed by gyrase and topoisomerase.
Figure 19.34: Transcription
generates more tightly wound DNA
ahead of RNA polymerase.
19.4 Bacterial RNA Polymerase Consists of
Multiple Subunits
• holoenzyme – The RNA polymerase form
that is competent to initiate transcription. It
consists of the five subunits of the core
enzyme and σ factor.
• Bacterial RNA core polymerases are ~400 kD
multisubunit complexes with the general
structure α2ββ′.
• Catalysis derives from the β and β′ subunits.
• CTD (C-terminal domain) – The domain of
RNA polymerase that is involved in
stimulating transcription by contact with
regulatory proteins.
Figure 19.07: Eubacterial RNA
polymerases have five types of
subunits.
19.4 Bacterial RNA Polymerase Consists of
Multiple Subunits
Figure 19.08: The upstream face of the core
RNA polymerase, illustrating the ‘crabclaw’
shape of the enzyme.
Figure 19.09: The structure of the RNA
polymerase core enzyme for the
bacterium Thermus aquaticus.
Adapted from K. M. Geszvain and R. Landick (ed. N. P.
Higgins). The Bacterial Chromosome. American Society for
Microbiology, 2004.
Structure from Protein Data Bank 1HQM. L.
Minakhin, et al., Proc. Natl. Acad. Sci. USA 98 (2001):
892-897.
19.5 RNA Polymerase Holoenzyme
Consists of the Core Enzyme and
Sigma Factor
• Bacterial RNA polymerase can
be divided into the α2ββ′ core
enzyme that catalyzes
transcription and the  subunit
that is required only for
initiation.
• Sigma factor changes the
DNA-binding properties of RNA
polymerase so that its affinity
for general DNA is reduced and
its affinity for promoters is
increased.
Figure 19.10: Core enzyme binds
indiscriminately to any DNA.
19.7 The Holoenzyme Goes Through
Transitions in the Process of Recognizing and
Escaping from Promoters
Figure 19.13: RNA polymerase initially contacts the region from –55 to +20.
19.7 The Holoenzyme Goes Through
Transitions in the Process of Recognizing and
Escaping from Promoters
Figure 19.12: RNA polymerase passes through several steps prior to elongation.
Adapted from S. P. Haugen, W. Ross, and R. L. Gourse,
Nat. Rev. Microbiol. 6 (2008): 507-519.
19.7 The Holoenzyme Goes Through
Transitions in the Process of Recognizing
and Escaping from Promoters
• ternary complex – The complex in initiation of
transcription that consists of RNA polymerase and DNA
as well as a dinucleotide that represents the first two
bases in the RNA product.
• There may be a cycle of abortive initiations before the
enzyme moves to the next phase.
• Sigma factor is usually released from RNA polymerase
when the nascent RNA chain reaches ~10 bases in
length.
19.8 Sigma Factor Controls Binding to DNA
by Recognizing Specific Sequences in
Promoters
• conserved sequence – Sequences in which many
examples of a particular nucleic acid or protein are
compared and the same individual bases or amino acids
are always found at particular locations.
• A promoter is defined by the presence of short
consensus sequences at specific locations.
19.8 Sigma Factor Controls Binding to DNA
by Recognizing Specific Sequences in
Promoters
• The promoter consensus sequences usually consist of a
purine at the start point, a hexamer with a sequence
close to TATAAT centered at ~ –10 (–10 element or
Pribnow box), and another hexamer with a sequence
similar to TTGACA centered at ~–35 (–35 element).
• Individual promoters usually differ from the consensus at
one or more positions.
19.8 Sigma Factor Controls Binding to DNA by
Recognizing Specific Sequences in Promoters
• The α subunit also contributes to promoter recognition.
• Promoter efficiency can be affected by additional elements as well.
• UP element – A sequence in bacteria adjacent to the promoter,
upstream of the –35 element, that enhances transcription.
Figure 19.14: DNA elements and RNA polymerase modules that contribute to promoter
recognition by sigma factor.
Adapted from S. P. Haugen, W. Ross, and R. L. Gourse, Nat. Rev.
Microbiol. 6 (2008): 507-519.
19.9 Promoter Efficiencies Can Be
Increased or Decreased by Mutation
• Down mutations to decrease promoter efficiency
usually decrease conformance to the consensus
sequences, whereas up mutations have the opposite
effect.
• Mutations in the –35 sequence can affect initial binding
of RNA polymerase.
• Mutations in the –10 sequence can affect binding or the
melting reaction that converts a closed to an open
complex.
19.10 Multiple Regions in RNA Polymerase
Directly Contact Promoter DNA
• The structure of σ70 changes when it associates with core enzyme, allowing
its DNA-binding regions to interact with the promoter.
• Multiple regions in σ70 interact with the promoter.
• The α subunit also contributes to promoter recognition.
Figure 19.17: Amino acids in the 2.4
-helix of σ70 contact specific bases in
the coding strand of the –10
promoter sequence.
Figure 19.18: The N-terminus of sigma blocks
the DNA-binding regions from binding to DNA.
Much greater discrimination between base pairs is possible
from the major groove side.
19.10 Multiple Regions in RNA Polymerase
Directly Contact Promoter DNA
Figure 19.16: The structure of sigma factor in the context of the holoenzyme:-10 and -35
interactions.
StructurefromProteinDataBank1IW7.D.
G.Vassylyev,etal.,Nature417(2002):712-
719.
Illustration adapted from D. G. Vassylyev, et al.,
Nature 417 (2002): 712-719.
19.11 RNA Polymerase–Promoter and DNA–
Protein Interactions Are the Same for Promoter
Recognition and DNA Melting
• footprinting – A technique
for identifying the site on
DNA bound by some protein
by virtue of the protection of
bonds in this region against
attack by nucleases.
Figure 19.21: Footprinting identifies DNA-binding sites
for proteins by their protection against nicking.
19.11 RNA Polymerase–Promoter and DNA–
Protein Interactions Are the Same for Promoter
Recognition and DNA Melting
• The consensus sequences at –35 and –10 provide most
of the contact points for RNA polymerase in the promoter.
• The points of contact lie primarily on one face of the DNA.
• Melting the double helix begins with base flipping within
the promoter.
Figure 19.22: One face of the promoter
contains the contact points for RNA.
19.12 Interactions
Between Sigma Factor
and Core RNA
Polymerase Change
During Promoter
Escape
Figure 19.24: Sigma factor and core
enzyme recycle at different points in
transcription.
19.12 Interactions Between Sigma Factor
and Core RNA Polymerase Change During
Promoter Escape
• A domain in sigma occupies the RNA exit channel and
must be displaced to accommodate RNA synthesis.
• Abortive initiations usually occur before the enzyme
forms a true elongation complex.
• Sigma factor is usually released from RNA polymerase
by the time the nascent RNA chain reaches ~10 nt in
length.
19.7 The Holoenzyme Goes Through
Transitions in the Process of Recognizing and
Escaping from Promoters
Figure 19.12: RNA polymerase passes through several steps prior to elongation.
Adapted from S. P. Haugen, W. Ross, and R. L. Gourse,
Nat. Rev. Microbiol. 6 (2008): 507-519.
26.8 lac Repressor Binding to the Operator
represses transcription simply by blocking
RNA polymerase binding to the lac promoter
• lac repressor protein binds to the double-stranded DNA
sequence of the operator.
• The operator is a palindromic sequence of 26 bp.
• Each inverted repeat of the operator binds to the DNAbinding
site of one repressor subunit.
Figure 26.17: The lac operator has a symmetrical sequence.
19.19 Replacement of/Competition for
Sigma Factors Can Regulate Initiation
• E. coli has seven sigma
factors, each of which causes
RNA polymerase to initiate at a
set of promoters defined by
specific –35 and –10
sequences.
Figure 19.36: The sigma factor associated with
core enzyme determines the set of promoters
at which transcription is initiated.
19.19 Replacement of/ Competition for
Sigma Factors Can Regulate Initiation
• The activities of the different sigma factors are regulated
by different mechanisms.
• anti-sigma factor – A protein that binds to a sigma
factor to inhibit its ability to utilize specific promoters.
Figure 19.37: In addition
to 70, E. coli has several
sigma factors that are
induced by particular
environmental
conditions.
19.19 Replacement of/ Competition for
Sigma Factors Can Regulate Initiation
• Heat-shock response – A set of loci that is
activated in response to an increase in
temperature that causes proteins to denature
(and other abuses to the cell).
– All organisms have this response.
– The gene products usually include chaperones that
act on denatured proteins.
19.15 Bacterial RNA Polymerase
Terminates at Discrete Sites
• There are two classes of terminators: Those
recognized solely by RNA polymerase itself
without the requirement for any cellular factors
are usually referred to as “intrinsic
terminators.”
– Others require a cellular protein called rho and are
referred to as “rho-dependent terminators.”
19.15 Bacterial RNA Polymerase
Terminates at Discrete Sites
Figure 19.28: The DNA sequences required
for termination are located upstream of the
terminator sequence.
19.15 Bacterial RNA Polymerase
Terminates at Discrete Sites
• Intrinsic termination requires recognition
of a terminator sequence in DNA that
codes for a hairpin structure in the
RNA product. The highly-transcribed
rRNA transcripts (rrn operon) and tRNA
operons terminate this way too.
• The signals for termination lie mostly
within sequences already transcribed
by RNA polymerase, and thus
termination relies on scrutiny of the
template and/or the RNA product that
the polymerase is transcribing.
• May require NusA protein that contacts
alpha subunit replacing replacing
sigma. NusA is the only other factor
conserved in eukaryotes.
Figure 19.29: Intrinsic terminators
include palindromic regions that
form hairpins varying in length
from 7 to 20 bp.
19.15 Bacterial RNA Polymerase
Terminates at Discrete Sites
• readthrough – It occurs at transcription or translation
when RNA polymerase or the ribosome, respectively,
ignores a termination signal because of a mutation of the
template or the behavior of an accessory factor.
• antitermination – A mechanism of transcriptional control
in which termination is prevented at a specific terminator
site, allowing RNA polymerase to read into the genes
beyond it. Lambda phage N and Q proteins.
19.16 How Does Rho Factor Work?
Figure 19.30: Rho factor binds to RNA at a rut
site and translocates along RNA until it reaches
the RNA–DNA hybrid in RNA polymerase.
• Rho factor is a protein that binds to
nascent RNA and tracks along the RNA
to interact with RNA polymerase and
release it from the elongation complex.
• rut – An acronym for rho utilization site,
the sequence of RNA that is recognized
by the rho termination factor.
19.16 How Does Rho Factor Work?
• Mutation polarity – The effect of a mutation in one gene
in influencing the expression (at transcription or
translation) of subsequent genes in the same transcription
unit.
Figure 19.33: The action of rho factor
may create a link between transcription
and translation.
Molecular Genetics 3 Lecture 5 4.10.11
Transcription in eukaryotes.
Eukaryotes, RNA polymerases and RNA pol. II promoters.
Transcription initiation by RNA pol. II.
Liam Keegan
MRC Human Genetics Unit, Western General Hospital, Edinburgh
Eukaryotic total RNA.
Ribosomal RNAs and tRNAs are major bands, mRNA is a
smear on denaturing gel stained with Ethidium Bromide.
• Eukaryotic RNA Polymerases
•
• Three nuclear RNA polymerases (E. coli has only one).
•
• RNA pol I (in nucleolus)
• transcribes rRNA genes → 50 – 70 % cell’s RNA synthesis
• resistant to > 500 g/ml -amanitin, an octapeptide from
• Amanita phalloides (Death Cap Mushroom) that grows near Oak trees.
•
• RNA pol II (in nucleoplasm)
• transcribes all protein-encoding genes & most small nuclear RNAs
• → 20 – 40% cell’s RNA synthesis
• inhibited by low ~ 0.03 g/ml -amanitin
•
• RNA pol III (in nucleoplasm)
• transcribes 5S, tRNA genes & some small nuclear RNAs
• → < 10% cell’s RNA synthesis
• inhibited by 20 g/ml -amanitin in animal cells
• resistant to -amanitin in yeast and insects
Eukaryotic RNA polymerases are similar to that of E. coli
but have 12 subunits.
20.2 Eukaryotic RNA Polymerases Consist
of Many Subunits
• All eukaryotic RNA
polymerases have ~12
subunits and are complexes
of ~500 kD.
• Some subunits are common
to all three RNA
polymerases.
• The largest subunit in RNA
polymerase II has a CTD
(carboxy-terminal domain)
consisting of multiple repeats
of a heptamer.
Figure 20.02: Some subunits are
common to all classes of eukaryotic RNA
polymerases and some are related to
bacterial RNA polymerase.
Archaebacteria have RNA polymerase with
eukaryote-like subunits.
J Biol Chem. 2006 Oct 13;281(41):30581-92. Epub 2006 Aug 1.
Protein-protein interactions in the archaeal transcriptional machinery: binding
studies of isolated RNA polymerase subunits and transcription factors.
Goede B, Naji S, von Kampen O, Ilg K, Thomm M.
Of the Eukaryotic genes that can be
traced back to such ancient origins
70% come from Eubacteria and 30%
come from Archaea.
The 30% that are most related to
Archaea include proteins involved in
nuclear processes.
Eukaryotic RNA polymerase subunits,
TATA-binding protein (TBP), TFIIB
and histones have Archaeal
homologs.
20.3 RNA Polymerase I Has a Bipartite
Promoter
Figure 20.03: Transcription units for RNA polymerase I have a core promoter separated by
~70 bp from the upstream promoter element.
20.3 RNA Polymerase I Has a Bipartite
Promoter
• SL1 includes the factor TATA-binding protein (TBP)
that is involved in initiation by all three RNA
polymerases.
• RNA polymerase I binds to the UBF1-SL1 complex at the
core promoter, or arrives already associated with it.
20.4 RNA Polymerase III Uses Downstream
and Upstream Promoters
• RNA polymerase III has two types of promoters.
• Internal promoters have short consensus sequences
located within the transcription unit and cause initiation
to occur at a fixed distance upstream.
• Upstream promoters contain three short consensus
sequences upstream of the start point that are bound by
transcription factors.
Figure 20.04: Promoters for RNA
polymerase III. 5S RNA, tRNAs,
snRNAs
20.4 RNA Polymerase III Uses Downstream
and Upstream Promoters
• assembly factors –
Proteins that are
required for formation
of a macromolecular
structure but are not
themselves part of
that structure.
• TFIIIA and TFIIIC bind
to the consensus
sequences and
enable TFIIIB to bind
at the startpoint.
Figure 20.05: Internal
type 2 pol III
promoters.
Figure 20.06: Internal
type 1 pol III
promoters.
20.4 RNA Polymerase III Uses Downstream
and Upstream Promoters
• TFIIIB has TBP as one subunit and enables RNA
polymerase to bind.
• preinitiation complex – The assembly of transcription
factors at the promoter before RNA polymerase binds in
eukaryotic transcription.
Transcription of protein-coding mRNAs
by RNA polymerase II.
Defining RNA polymerase II
promoters.
• The TATA box is a common component of RNA polymerase II promoters
– It consists of an A-T-rich octamer located ~25 bp upstream of the startpoint.
• The DPE is a common component of RNA polymerase II promoters that do not contain a TATA box.
• A core promoter for RNA polymerase II includes:
– the InR
– either a TATA box or a DPE
Figure 24.10
Promoters for protein-coding transcripts.
• hnRNA/pre-mRNA processing
• A typical eukaryotic mRNA:
•
• 5'cap..............________________________________.....................AAAAAAAAAn
• nontranslated coding long 3' trailer n =100-200
• leader <300nt often >1000nt
7-methyl-Guanosine cap and polyA tail protect eukaryotic mRNA
against exonucleases and make it much more long-lived than
prokaryotic mRNA (Lecture 7).
polyA+ RNA purification and old fashioned cDNA synthesis
Second strand initiation depended on random hairpin ends on first strand cDNA. Inefficient.
Newer methods of cDNA synthesis use random primers to initiate second strand synthesis.
Mapping transcripts and transcription
start sites.
PolyA and Cap uses in cDNA cloning.
• Especially in the early days cDNA synthesis using oligo-dT primer and reverse
transcriptase allowed cDNA clones to be obtained that covered most of the
transcript sequence but cDNA clones often failed to reach 5’ end of transcript.
• Ribo depletion is used instead of poly A + RNA isolation now. Ribodepletion uses
beads with probes homologous to rRNA to remove ribosomal RNA from total
RNA for sequencing. Nowadays, getting full length cDNA is sometimes improved
by using an antibody to Cap to enrich in full length mRNAs first. (Newer Capselected
cDNA libraries for genome sequencing projects.)
• Next Generation Sequencing is now completing analysis of transcriptomes (total
genome-wide RNA sets). To see where RNA-binding proteins are located RNA
immunoprecipitation seq (Rip Seq, like ChIp Seq for DNA-binding proteins)) is
used.
General transcription factors are
required by RNA polymerase II to
recognize promoters.
24.7 Accurate initiation by RNA Polymerase II
in S100 nuclear extracts in vitro.
• Purified RNA polymerase would not initiate transcription
correctly at a promoter on a DNA fragment without additional
factors. It would start at the end of a linearized DNA fragment
and transcribe from there. Adding an S100 nuclear extract
allowed accurate initiation at promoters. First nuclei are
purified from cultured human HeLa cells on a sucrose density
gradient. Then nuclei are lysed and a clear supernatant
obtained after 100,000 G centrifugation.
• RNA polymerase II requires general transcription factors (called
TFIIX) to recognize promoters and to initiate transcription at the
correct starting site.
Radiolabeled runoff transcripts show accurate initiation of
new transcripts at promoter in nuclear extract in vitro.
•
Primer extension analysis was also used to check accuracy
of start and levels of transcription in transfected cells in vivo.
A nuclear extract from HeLa cells that gave accurate
initiation at Pol II promoters was fractionated by ionexchange
column chromatography.
- Charge on column
+ charge on column
+ charge on column
Increasing salt concentration
TATA-binding protein (TBP) and 12 TBP associated factors (TAFs) comprise
TFIID.
TFIIB binds asymmetrically and sets direction of transcription.
Transcription start.
24.10 The Basal Apparatus Assembles at the
Promoter. General Transcription Factors.
• Binding of TFIID to the TATA box is the first step in
initiation.
• Other transcription factors bind to the complex in
a defined order
– This extends the length of the protected region
on DNA (by DNAse1 footprinting).
• When RNA polymerase II binds to the complex, it
may initiates transcription.
• Key step is promoter clearance by pol II after
initiation
Figure 24.14
24.11 Initiation Is Followed by Promoter Clearance of
RNA polymerase II
• TFIIE and TFIIH are required to melt DNA and
move with polymerase.
• Capping and Ser2 and Ser5 Phosphorylation
of the CTD may be required for elongation to
begin.
• Many promoters have polymerase II paused,
waiting for a further signal (e.g. Drosophila
Hsp70 promoters)
• The CTD may coordinate processing of RNA
with transcription. Recruit 3’ end processing
and splicing factors.
Figure 24.17
Figure 02.01: Each chromosome has a single long molecule of DNA within which are the
sequences of individual genes.
‘Compare and contrast’
transcription in prokaryotes and eukaryotes
2.2 Most Genes Encode Polypeptides
• heteromultimer – A molecular complex (such as a
protein) composed of different subunits.
• homomultimer – A molecular complex (such as a
protein) in which the subunits are identical.
• Alphafold2, (Google DeepMind, 2021) is a neural
network type, machine learning algorithm trained on
known protein structures to predict new protein
structures accurately. Multiple sequence alignments
(MSAs) show evolutionary covariation of amino acid
changes and allow a protein sequence to be assigned to
one of over a thousand domain structure families.
Further structure refinement is based on side chain
packing.