Lecture 20 DNA Repair and Genetic Recombination
(Chapter 16 and Chapter 15 Genes X)
Exert from Darwin’s diary
20
Exert from Darwin’s diary
22
• gene families – sets of genes within a genome that
code for related or identical proteins or RNAs.
– The members were derived by duplication of an ancestral gene followed by
accumulation of changes in sequence between the copies.
– Most often the members are related but not identical.
• pseudogenes – Inactive but stable components of the
genome derived by mutation of an ancestral active
gene.
– Usually they are inactive because of mutations that block transcription or
translation or both.
• gene cluster – A group of adjacent genes that are
identical or related.
Gene Duplication Major Force in Genome Evolution
After a globin gene has been duplicated, differences may accumulate
between the copies
A simple model of mutational change in which alpha is the probability of a
transition and beta is the probability of a transversion
Reproduced from MEGA (Molecular Evolutionary Genetics Analysis) by S. Kumar,
K. Tamura, and J. Dudley. Used with permission of Masatoshi Nei, Pennsylvania
State University.
• The probability of a mutation is influenced by the likelihood
that the particular error/change will occur and the likelihood
that it will be repaired.
• synonymous mutation – A change in DNA sequence in a
coding region that does not alter the amino acid that is
encoded…. potentially no selective pressure
• nonsynonymous mutation – A change in DNA sequence in
a coding region that alters the amino acid that is encoded….
increases the likelihood of selective pressure.
• Neutral muta*on -‐a change in DNA sequence that gives no selec4ve advantage or disadvantage
Selection Can Be Detected by Measuring
Sequence Variation
• The ratio of non synonymous to synonymous
substitutions in the evolutionary history of a gene is a
measure of positive or negative selection.
• Low heterozygosity of a gene may indicate recent
selective events.
• genetic hitchhiking – The change in frequency of a
genetic variant due to its linkage to a selected variant at
another locus.
DNA Sequences Evolve by Mutation followed
by some some form of “Sorting Mechanism”
• In small populations, the frequency of a mutation will
change randomly and new mutations are likely to be
eliminated by chance.
• fixation – The process by which a new allele replaces the
allele that was previously predominant in a population.
• The frequency of a neutral mutation largely depends on
genetic drift, the strength of which depends on the size of
the population.
• The frequency of a mutation that affects phenotype will be
influenced by negative or positive selection.
The fixation or loss of alleles by random genetic drift occurs more rapidly in
(A) populations of 10 than in (B) populations of 100
Data courtesy of Kent E. Holsinger, University of Connecticut [http://
darwin.eeb.uconn.edu]
• Comparing the rates of substitution among related species
can indicate whether selection on the gene has occurred.
• linkage disequilibrium – A nonrandom association
between alleles at two different loci, often as a result of
linkage.
A higher number of non synonymous substitutions in lysozyme sequences in
the cow/deer lineage as compared to the pig lineage
Adapted from N. H. Barton, et al. Evolution. Cold Spring Harbor Laboratory Press,
2007. Original figure appeared in J. H. Gillespie, The Causes of Molecular
Evolution. Oxford University Press, 1991.
Selection Can Be Detected by Measuring
Sequence Variation
The recently cloned G6PD allele has rapidly increased in
frequency though positive selection -the allele confers
some degree of resistance to malaria
Adapted from E. T. Wang, et al., Proc. Natl. Acad. Sci. USA 103 (2006): 135-140.
A Constant Rate of Sequence Divergence Is a
Molecular Clock
• The sequences of orthologous genes in different species
vary at non synonymous sites (where mutations have
caused amino acid substitutions) and synonymous sites
(where mutations have not affected the amino acid
sequence).
• Synonymous substitutions accumulate ~10× faster than
non synonymous substitutions, but the rate of change
appears to be similar.
A Constant Rate of Sequence Divergence Is a Molecular
Clock
Divergence of DNA sequences depends on evolutionary separation
Figure: the rate of evolution of hemoglobin. Each point on the graph is for a pair of species, or groups of
species. Some of the points are for a-hemoglobin, others for ß -hemoglobin. From Kimura (1983).
68
A Constant Rate of Sequence Divergence Is a Molecular
Clock
The rate of evolution of three types of proteins over time
Reproduced with kind permission from Springer Science+Business Media: J. Mol. Evol., The structure of cytochrome and the rates of
molecular evolution, vol. 1, 1971, pp. 26-45, R. E. Dickerson, fig. 3. Courtesy of Richard Dickerson, University of Californi
• The evolutionary divergence between two DNA
sequences is measured by the “corrected” percent of
positions at which the corresponding nucleotides differ.
• Substitutions may appear to accumulate (and become
incorporated into the populations gene pool) at a more or
less constant rate after genes separate, so that the
divergence between any pair of globin sequences (for
example) is proportional to the time since they shared a
common ancestry.
Rates of evolution for “meaningful “ (i.e. amino acid changing) and silent base changes in various
genes.
Rates are expressed as inferred number of base changes per 109 years. Simplified from
Li, Wu & Luo (1985).
72
“Confounding factors” in discerning “true” evolutionary changes......:
Multiple changes in DNA sequence at the same locus.
Population Size
Horizontal Gene Transfer.
Rate of Replications
Gene Duplication and Genome duplications
• Two genes are said to be orthologous if they diverged after a speciation event.
• Two genes are said to be paralogous if they diverged after a duplication event.
• The frequency of a neutral mutation largely depends on
genetic drift, the strength of which depends on the size of
the population.
• The frequency of a mutation that affects phenotype will be
influenced by negative or positive selection.
The fixation or loss of alleles by random genetic drift occurs more rapidly in
(A) populations of 10 than in (B) populations of 100
Data courtesy of Kent E. Holsinger, University of Connecticut [http://
darwin.eeb.uconn.edu]
“Confounding factors” in discerning “true” evolutionary changes......:
Multiple changes in DNA sequence at the same locus.
Population Size
Horizontal Gene Transfer.
Rate of Replications
Gene Duplication and Genome duplications
• Two genes are said to be orthologous if they diverged after a speciation event.
• Two genes are said to be paralogous if they diverged after a duplication event.
A
B
root
C
Warfarin works by acting against vitamin K. This vitamin
activates a number of genes that create clots in blood, but it
itself has to be activated by a protein called VKORC1.
Warfarin stops VKORC1 from doing its job, thereby
suppressing vitamin K. The clotting process fails, and
bleeds continue to bleed.
Rodents can evolve to shrug off warfarin by tweaking their
vkorc1 gene, which encodes the protein of the same
name. In European house mice, scientists have found at
least 10 different genetic changes (mutations) in vkorc1
that change how susceptible they are to warfarin. But only
six of these changes were the house mouse’s own
innovations. The other four came from a close relative – the
Algerian mouse, which is found throughout northern
Africa, Spain, Portugal, and southern France.
The two species separated from each other between 1.5 and
3 million years ago. They rarely meet, but when they do,
they can breed with one another. The two species have
identifiably different versions of vkorc1. But Song found
that virtually all Spanish house mice carry a copy of
vkorc1 that partially or totally matches the
Algerian mouse version. Even in Germany, where the
two species don’t mingle, a third of house mice carried
copies of vkorc1 that descended from Algerian peers.
“Confounding factors” in discerning “true” evolutionary changes......:
Multiple changes in DNA sequence at the same locus.
Population Size
Horizontal Gene Transfer.
Rate of Replications…..
Gene Duplication and Genome duplications
• Two genes are said to be orthologous if they diverged after a speciation event.
• Two genes are said to be paralogous if they diverged after a duplication event.
31
Some of the clusters of β-‐globin genes and pseudogenes that are found in vertebrates.
Diﬀerent hemoglobin genes are expressed during embryonic, fetal, and adult periods of human development.
Gene Duplication Provides a Major Force in Evolution of
the different genomes
• Most of the genes that are unique to vertebrates are
concerned with the immune or nervous systems.
• Duplicated genes may diverge to generate different
genes, or one copy may become an inactive pseudogene.
Gene Duplication Major Force in Genome Evolution
After a globin gene has been duplicated, differences may accumulate
between the copies
sequence1 sequence 2
(functional) (functional)
sequence 3
(pseudogenes)
84
Curiously, pseudogenes overall evolve at about the same rate as silent base changes. Rates
are expressed in numbers of base changes per 109 years. The comparisons are for various genes
and pseudogenes in the globin gene family.
Simplified from Li, Tanimura & Sharp (1987)
These changes include ALL genes, previous ccomparisons only related WELL established genes…..
Of course one should really take into account some of the additional “unseen factors that might
87
affect selective pressure on “silent” mutations.
85
Frequencies of six arginine codons in the DNA of three species.
The table gives the percentages of arginine amino acids that are encoded by each of the six
codons in various numbers of genes in species.
Simplified from Grantham, Perrin & Mouchiroud (1986).
86
122
http://home.planet.nl/~gkorthof/kortho51.htm
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125
How Did Interrupted Genes Evolve?
• A major evolutionary question is whether genes
originated with introns or whether they were
originally uninterrupted.
• “introns late” model – The hypothesis that the
earliest genes did not contain introns, and that
introns were subsequently added to some genes.
How Did Interrupted Genes Evolve?
• Interrupted genes that correspond either to
proteins or to independently functioning
nonprotein-encoding RNAs probably originated in
an interrupted form (the “introns early”
hypothesis).
• exon shuffling – The hypothesis that genes have
evolved by the recombination of various exons
coding for functional protein domains.
An exon surrounded by flanking sequences that is translocated into an
intron may be spliced into the RNA product
Gene Expression Prokaryotes Chapters 19, Genes X 48
49
50
• 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 DNA-dependent RNA
polymerase, DDRP).
FIGURE 01: One strand of
DNA is transcribed into RNA
Promoters and terminators
define the transcriptional unit
• upstream – Sequences that lie ahead of the defined
transcriptional unit • downstream – Sequences that extend farther in the direction
of expression within or after the transcription unit. • primary transcript – The original unmodified RNA product
corresponding to a transcription unit.
• nascent RNA – A ribonucleotide chain that is still being
synthesized, so that its 3' end is paired with DNA where RNA
polymerase is elongating. • monocistronic mRNA – mRNA that encodes one protein. • A bacterial mRNA may be (often is) polycistronic in having
several coding regions that represent different genes.
Transcription Occurs by Base Pairing in a
“Bubble” of Unpaired DNA
• RNA polymerase separates the two strands of DNA in a
transient “bubble” and uses one strand as a template to direct
synthesis of a complementary sequence of RNA.
• The bubble extends between 12 to 16 bp, and the RNA–DNA
hybrid within the bubble is 8 to 9 bp.
Transcription Occurs by Base Pairing in a “Bubble” of
Unpaired DNA
RNA polymerase surrounds the bubble
RNA synthesis occurs in the transcription bubble
•
RNA polymerase separates the two
strands of DNA in a transient “bubble” and
uses one strand as a template to direct
synthesis of a complementary sequence of
RNA.
•
The bubble is 12 to 14 bp, and the RNA–
DNA hybrid within the bubble is 8 to 9 bp.
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
core ~400 kD multisubunit
complexes with the general
structure α2ββ′ω.
FIGURE 07: RNA polymerase
has 4 types of subunit
Bacterial ω, archaeal RpoK, and eukaryotic RPB6 are sequence homologs.
Minakhin L et al. PNAS 2001;98:892-897
©2001 by National Academy of Sciences
Bacterial ω, archaeal RpoK, and eukaryotic RPB6 are sequence homologs. Aligned sequences of bacterial RNAP ω (Top), archaeal RNAP
RpoK (Middle), and poxviral and eukaryotic RNAP RPB6 (Bottom). Residues identical in at least half of the aligned sequences and
represented in all three sets of aligned sequences are in red; residues identical or similar in at least half of the aligned sequences and
represented in all three sets of aligned sequences are in blue. CR1–CR3 (yellow bars) delineate conserved regions (defined as containing
residues identical or similar in at least half of the aligned sequences and represented in all three sets of aligned sequences, and containing
no insertions or deletions greater than one residue). Helices 2 and 3 and strand 1 in the crystallographic structure of Thermus aquaticus ω
(Fig. 3) are indicated by black bars. Species names and database locus identifiers for the sequences are, in order:
56
The upstream face of the core RNA
polymerase
Adapted from K. M. Geszvain and R. Landick (ed. N. P. Higgins). The
Bacterial Chromosome. American Society for Microbiology, 2004.
The structure of RNA polymerase
looking through the main channel
Structure from Protein Data Bank 1HQM. L. Minakhin, et al., Proc.
Natl. Acad. Sci. USA 98 (2001): 892-897.
RNA Polymerase “Holoenzyme” Consists of the Core
Enzyme and then the Sigma Factor
Sigma factor controls specificity
•
Bacterial RNA polymerase can be
divided into the α2ββ′ω core enzyme
that catalyzes transcription and the σ
subunit that is required only for
initiation.
•
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.
•
The Sigma factor changes the DNAbinding properties of RNA polymerase
so that its affinity for general DNA is
reduced and its affinity for promoters is
increased.
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.
•
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 upstream from -and within the
transcriptional “unit”.
RNA polymerase changes size at
initiation
Sigma and core enzyme must
dissociate
• The rate at which RNA polymerase binds to promoters can be too
fast to be accounted for by simple diffusion.
• RNA polymerase binds to random sites on DNA and exchanges them
with other sequences until a promoter is found.
Proposed mechanisms for how RNA polymerase finds a promoter
Adapted from C. Bustamante, et al., J. Biol. Chem. 274 (1999):
166665-166668.
63
66
The Transcription Reaction Effectively Has Four Stages
RNA polymerase catalyzes transcription
•
RNA polymerase binds to a promoter site on DNA to
form a closed complex (I). •
When RNA polymerase binds to a promoter, it separates
the DNA strands to form a transcription bubble and
incorporates nucleotides into RNA.
•
RNA polymerase initiates transcription (initiation) after
opening the DNA duplex to form a transcription bubble
(the open complex) (II).
•
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.
•
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. (III)
•
Transcription stops (termination) and the DNA duplex
reforms when RNA polymerase dissociates at a
terminator site. (IV)
RNA polymerase actually 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.
Supercoiling Plays a Major Role in Transcription
•
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.
Transcription changes DNA structure
Consensus Promoter Sequences
–
•
-35
T82 T84 G78 A65 C54 a45
-10
<--- 17 bp ----> T80 A95 T45 A60 a50 T96
•
The promoter consensus sequences usually consist of a purine at the start point,
a hexamer with a sequence close to TATAAT centred at ~ –10 (–10 element or
TATA box), and another hexamer with a sequence similar to TTGACA centred at
~ –35 (–35 element).
•
Individual promoters usually differ from the consensus at one or more positions.
68
76
Promoter Efficiencies Can Be Increased or Decreased by
Mutation
•
Down mutations tend to decrease promoter efficiency, usually decrease conformance to the
preferred interactions with the “consensus sequences”, whereas up mutations have the
opposite effect.
•
Mutations in the –35 sequence tend to affect initial binding of RNA polymerase.
•
Mutations in the –10 sequence tend to affect binding of the holoenzyme or the melting
reaction that converts one of the closed complexes to an open complex. Sigma Factor Controls Binding to DNA by
Recognizing Specific Sequences in Promoters
• 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.
-DNA elements and the RNA polymerase modules contributing to promoter
recognition by sigma factor
Adapted from S. P. Haugen, W. Ross, and R. L. Gourse, Nat. Rev.
Microbiol. 6 (2008): 507-519.
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 RNA Polymerase Directly Contact
Promoter DNA
• Multiple regions in σ70 interact with the promoter.
• The α subunit also contributes to promoter recognition.
The 2.4 helix of sigma determines specificity
Sigma N-terminus controls DNA-binding
The structure of sigma factor
Structure from Protein Data Bank 1IW7. D. G. Vassylyev, et al., Nature 417
(2002): 712-719. Illustration adapted from D. G. Vassylyev, et al., Nature 417
(2002): 712-719.
Footprinting Is a High Resolution Method for Characterizing
RNA Polymerase–Promoter and DNA–Protein Interactions in
General
•
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.
A protein protects a series of bonds
against nuclease attack
Footprinting Is a High Resolution Method for Characterizing
RNA Polymerase–Promoter and DNA–Protein Interactions in
General
• 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.
RNA polymerase primarily contacts
one face of DNA
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. A Model for Enzyme Movement Is Suggested by the Crystal
Structure
• DNA moves through a channel in RNA polymerase and makes a sharp turn
at the active site.
• Changes in the conformations of certain flexible modules within the enzyme
control the entry of nucleotides to the active site. DNA turns as it is moved through the
active site
79
96
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.”
Bacterial termination occurs at
a discrete site
• Intrinsic termination requires the
recognition of a terminator sequence
in DNA that codes for a hairpin
structure in the RNA product.
• The signals for termination lie mostly
within sequences that have already
been transcribed by RNA
polymerase, and thus termination
relies on scrutiny of the template
and/or the RNA product that the
polymerase is transcribing.
FIGURE 28: An intrinsic
terminator has two features
100
83
• read through – Does occur at transcription or translation termination sites
when RNA polymerase or the ribosome, respectively, ignores a termination
signal because of a mutation of the template or the behaviour 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.
• polarity – The effect of a mutation in one gene influencing the expression
(though either transcription or translation) of subsequent genes in the same
transcription unit.
Competition for Sigma Factors Can Regulate Initiation
•
E. coli has several sigma factors, each of
which causes RNA polymerase to initiate
at a series of discrete promoters defined
by specific –35 and –10 sequences.
Sigma controls promoter
recognition
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.
E. coli has several sigma factors
Competition for Sigma Factors Can Regulate
Initiation
• heat shock response – A set of genetic loci that is
activated in response to an increase in temperature
that may otherwise cause 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.
Sigma Factors May Be Organized into Temporal
Cascades
•
A cascade of sigma factors is created
when one sigma factor is required to
transcribe the gene coding for the next
sigma factor.
•
The early genes of phage SPO1 are
transcribed by host RNA polymerase.
•
One of the early genes codes for a sigma
factor that causes RNA polymerase to
transcribe the middle genes. •
Two of the middle genes code for
subunits of a sigma factor that cause
RNA polymerase to transcribe the late
genes.
Alternative sigmas control phage
development
Sporulation Is Controlled by Sigma Factors
Sporulation occurs through an ordered series of sigma
production ordered events
Consensus Promoter Sequences
–
•
-35
T82 T84 G78 A65 C54 a45
-10
<--- 17 bp ----> T80 A95 T45 A60 a50 T96
90
FIGURE 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.
Regula(on of Transcrip(on in prokaryotes is a complex and mul(-‐(ered phenomenon.
• RNA polymerase -‐Sigma interac*ons -‐dictate where the RNA polymerase binds….. • Organiza4on of gene Clusters…. rela4ve to the origin of replica4on… and to each other.
93
Regula(on of Transcrip(on in prokaryotes is a complex and mul(-‐(ered phenomenon.
• Organiza4on of gene Clusters…. rela4ve to the origin of replica4on… and to each other. • example, the lac operon.
94
• In negative regulation, a repressor protein binds to an operator to
prevent a gene from being expressed.
• In positive regulation, a transcription factor is required to bind at the
promoter in order to enable RNA polymerase to initiate transcription.
A repressor stops RNA polymerase from
initiating
Transcription factors enable RNA polymerase to bind to the
promoter
Contacts can be enhanced by proxy…...
96
83
Regula(on of Transcrip(on in prokaryotes is a complex and mul(-‐(ered phenomenon.
• RNA polymerase -‐Sigma interac*ons -‐dictate where the RNA polymerase binds….. • Organiza4on of gene Clusters…. rela4ve to the origin of replica4on… and to each other.
97
Regula(on of Transcrip(on in prokaryotes is a complex and mul(-‐(ered phenomenon.
98
99
lacZ promoter -‐loss of consensus: op4mal expression NOT maximal expression
–
•
-35
T82 T84 G78 A65 C54 a45
-10
<--- 17 bp ----> T80 A95 T45 A60 a50 T96
100
The lac Operon Has a Second Layer of Control: Catabolite
Repression
• A dimer of CAP (sometimes called
CRP) is activated by a single
molecule of cyclic AMP (cAMP).
• cAMP is controlled by the level of
glucose in the cell; a low glucose
level allows cAMP to be made.
• CAP or CRP interacts with the Cterminal domain of the α subunit of
RNA polymerase to activate it.
FIGURE 27: Glucose reduces CRP activity
102
20
• We can combine all activation
and repressible activities in to
four distinct combinations:
• negative inducible,
• negative repressible,
• positive inducible, and
• positive repressible.
Induction and repression can be under positive or
negative control
hYp://biotech.gsu.edu/
houghton'04/
Regulatory_models.html
104
105
15
Structure from Protein Data Bank 1LBG. M. Lewis, et al., Science 271 (1996):
1247-1254. Photo courtesy of Hongli Zhan and Kathleen S. Matthews, Rice
University.
FIGURE 13: Lac repressor monomer has several
domains
• Different types of mutations
occur in different domains of
the repressor protein.
Mutations identify repressor domains
lac Repressor Binding to the Operator Is Regulated by
an Allosteric Change in Conformation
• Inducer binding causes a change
in repressor conformation that
reduces its affinity for DNA and
releases it from the operator.
FIGURE 18: Inducer controls repressor
conformation
• Monomers form a dimer by
making contacts between core
subdomains 1 and 2.
• Dimers can also form a tetramer
by interactions between the
tetramerization helices.
• Bipartite nature of the lac
repressor
FIGURE 15: Repressor is a tetramer of two dimers
• Each dimer in a repressor tetramer can bind an operator, so that the
tetramer can bind two operators simultaneously.
• Full repression requires the repressor to bind to an additional
operator downstream or upstream as well as to the primary operator
at the lacZ promoter.
• Binding of repressor at the operator stimulates binding of RNA
polymerase at the promoter but precludes transcription.
Repressor can make a loop in DNA
The Operator Competes with Low-Affinity Sites to Bind
Repressor
•
Proteins that have a high affinity for a specific DNA sequence also have a
low affinity for other DNA sequences.
•
Every base pair in the bacterial genome is the start of a low-affinity binding
site for repressor.
Repressor specifically binds operator DNA
The Operator Competes with Low-Affinity Sites to Bind
Repressor
• The large number of lowaffinity sites ensures that all
repressor protein is bound to
DNA.
• Repressor binds to the
operator by moving from a lowaffinity site rather than by
equilibrating from solution.
FIGURE 24: Repression affects the sites at which
repressor is bound on DNA
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18
• Binding of repressor at the operator stimulates binding of RNA
polymerase at the promoter but precludes transcription.
• It also opens up the “activator” site for binding of CAP the “Catabolite
Activator Protein” to bind…...and as soon as lactose is present the
system is primed to go!!!
FIGURE 21: Repressor can make a loop in DNA
115
23
• We can combine all activation
and repressible activities in to
four distinct combinations:
• negative inducible,
• negative repressible,
• positive inducible, and
• positive repressible.
Induction and repression can be under positive or
negative control
117
118
119
55
Transcriptional Termination Can Also
Be a Regulatory Event
Rho terminates
transcription
How Does Rho Factor Work?
• 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.
• 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.
• antitermination complex – Proteins that allow RNA polymerase
to transcribe through certain terminator sites.
Rho can terminate when a
nonsense mutation removes
ribosomes
Antitermination Can Be a Regulatory Event
• An antitermination complex allows RNA polymerase to read
through terminators.
Action at a terminator controls transcription
Antitermination Can Be a Regulatory Event
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6
125
7
126
127
8
128
9
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.
E. coli has several sigma factors
Alternative Regulatory Mechanisms Through Alternative
Sigma Factors…….
The mode of control of sigma54 (the gene product of ntrA or rpoN) is achieved, because
(unlike sigma70) sigma54 cannot function alone -it requires interaction with another
protein NtrC (NRI), which is the gene product of the ntrC gene. Moreover, it is not just the
NtrC (NRI) that is required, because NRI has to be activated into NRI -phosphate by
becoming phosphorylated.
NRI is a DNA binding protein which, when phosphorylated binds to specific sequences of
DNA and confers initiation activity on sigma54, promoting the polymerase's ability to form
the Rpol/promoter "open complex". These binding sites do not have to be proximal to
the promoter...protein interactions at a distance!!!
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The mode of control of sigma54 (the gene product of ntrA or rpoN) is achieved, because
(unlike sigma70) sigma54 cannot function alone -it requires interaction with another
protein NtrC (NRI), which is the gene product of the ntrC gene. Moreover, it is not just the
NtrC (NRI) that is required, because NRI has to be activated into NRI -phosphate by
becoming phosphorylated.
NRI is a DNA binding protein which, when phosphorylated binds to specific sequences of
DNA and confers initiation activity on sigma54, promoting the polymerase's ability to form
the Rpol/promoter "open complex". These binding sites do not have to be proximal to
the promoter...protein interactions at a distance!!!
The question now is how does NRI become phosphorylated? Through the action of NRII
of course, which is a kinase that responds to levels of NH4+ in the cell
Herein, finally lies the connection between specific transcriptional initiation factors and
levels of nitrogen in the cell.
NRII is the gene product of ntrB (glnL in E. coli), and relates to ntrC in that it is a member
of the same operon -as is glutamine synthetase (glnA), which is responsible for
converting glutamate into glutamine in the presence of NH4+.
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Eukaryotic Transcription.... Similar Themes, But a
Little Different
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hYp://biotech.gsu.edu/
houghton'04/
Regulatory_models.html
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