BIO311 Prokaryote Gene Expression Section 2 Transcription of RNA

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BIO311 - Prokaryote Gene ExpressionSection
2Transcription of RNA

• Prof Jasper Rees
• Department of Biotechnology, UWC
• www.biotechnology.uwc.ac.za/teaching/BIO311

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Overview - Section 2

• The structure of transcription units
• Catalysis of RNA synthesis by RNA polymerase
• Structure of RNA polymerases
• Role of Sigma factors in Initiation and promoter
  selection
• Kinetics and Thermodynamics of promoter selection
• Promoter structure
• RNA Polymerase interactions with DNA
• Termination - rho dependent and rho independent
• Anti termination factors

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RNA synthesis
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Transcription units

• The region of DNA which is transcribed into RNA
  by RNA Polymerase
• From Promoter at the 5 end
• To the Terminator at the 3 end

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RNA Transcription process

• Initiation of synthesis - promoter recognition by
  RNA polymerase and regulatory protein factors
• Elongation phase - synthesis of RNA
• Termination - terminator recognition and end of
  elongation cycle and disociation of RNA and
  polymerase from DNA

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Transcription Bubbles

• Transcription requires the denaturation of the
  DNA template
• This reveals the template strand of the DNA
• This is then copied into the complementary sense
  strand of RNA

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RNA Polymerase activities

• Promoter recognition
• Promoter denaturation (melting)
• DNA unwinding
• Single stranded DNA binding sites
• Synthesis of RNA
• Melting of RNA from template DNA
• Double stranded DNA rewinding
• Termination site recognition and termination of
  RNA synthesis

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RNA Polymerase activities
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Yeast RNA Polymerase

• Low resolution (7 Å) structure shown here
• Binding sites for DNA and mRNA modelled
• More recent structures now available

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RNA polymerisation reaction
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RNA synthesis - elongation cycle

• Cycle of synthesis during elongation involves
  synthesis of 2-9 bases, with compression of RNA
  polymerase at the rear
• Then rapid movement of front of enzyme

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Mechanism of compression?

• The mechanism of the compression - extension
  cycle of the RNA polymerase in unknown
• It must involve significant changes in the
  conformation of the enzyme
• At present 3D structure data does not reveal this
  mechanism
• Role of the mechanism in unknown

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Prokaryote RNA Polymerases
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Role of Sigma (s? factor

• Sigma is required for formation of Holoenzyme of
  RNA Polymerase
• Its role is in the selection of promoter
  sequences at which Inititation complexes will be
  formed by the RNA Pol
• It is a sequence specific DNA binding protein
• It can only bind DNA when part of the RNA
  Polymerase holoenzyme
• Role is to direct the binding of RNA Polymerase
  to promoter sequences

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Core Enzyme and Holoenzyme

• RNA polymerase found in two forms
• Core enzyme made up of a2bb
• Holoenzyme made up of a2bbs
• Holoenzyme required for promoter binding
• Core enzyme catalyses RNA synthesis

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Initiation complexes

• Holoenzyme binds to promoter sequence in
  Initiation complex
• Sigma is dissociated and contacts with promoter
  sequence released
• RNA Pol Core Enzyme remains bound initial
  elongation complex
• General Elongation complex forms and RNA
  synthesis occurs

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Formation of initiation complex

• Described by thermodynamic and kinetic parameters
• Some steps are effectively irreversible
• Overall process is rate limited by rate of
  release of sigma factor

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Binding of s and RNA Pol to DNA

• Sigma cannot bind DNA alone (N terminal domain
  blocks this)
• Core enzyme and holoenzyme can both bind to DNA
  at non-specific sites
• Holoenzyme binding to specific sites (promoters)
  is caused by sigma - which reduces the affinity
  for general DNA binding compared with the core
  enzyme
• Core enzyme engaged in transcription is in
  complexes with DNA (not bound to specific
  sequences)
• There is little (or no) free RNA polymerase in
  the cell, because it all of the holoenzyme and
  core enzyme is complexed with DNA

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Distribution of RNA Pol on DNA

• Core enzyme and Holoenzyme in loose complexes is
  bound non-specifically to DNA
• Holoenzyme in tight (closed or open) complexes is
  bound to promoter sequences
• Ratio of loose to tight complex shows that
  binding is approximately 5000 times tighter at
  promoters

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Rapid exchange of RNA Pol on DNA drive promoter
selection

• Three possible models for selection of promoter
  sequences
• Kinetic data shows process is faster than random
  diffusion
• Sliding along DNA does not occur
• Therefore mechanism involving strand exchange
  must be occurring
• This allows very rapid location of promoter
  sequences

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Sigma recycling allows promoter selection and
then elongation

• Sigma provides high affinity binding for
  promoters, and thus selectivity
• When sigma dissociates, core enzyme still binds
  to DNA but without sequence specificity, allowing
  elongation to occur without premature termination

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Role of s in abortive initiation

• Abortive initiation is the process where the
  Holoenzyme synthesises 2-9 oligonucleotides while
  bound to the promoter
• This occurs until sigma is released and
  elongation proceeds
• Rate of release of sigma determines the rate of
  synthesis of RNA molecules from a promoter
• Minimum release time for sigma is approximately 1
  second
• Thus maximum rate of synthesis is approximately 1
  RNA/second for any promoter
• Minimum rate of synthesis is as low as 1RNA/hour

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s factor drives selection of promoters

• The binding of the holoenzyme to the promoter
  sequences is efficient
• 50-100 of all promoters have RNA Pol bound at
  any one time (since E.coli has about 5000 genes
  in approximately 1000 operons)
• Many promoters are under repression control, so
  that expression can occur as soon as the
  repressor is released
• Even repressed genes will be expressed at a very
  very low rate (thermodynamic requirement!)
• Low rate of expression often required for
  biological control and response (as with the lac
  operon)

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Promoters are defined by consensus sequences

• A consensus sequence is defined by the frequency
  matrix of a set of aligned sequences (whether
  DNA, RNA or Protein)
• By aligning promoter sequences we can identify
  conserved positions
• These are found in many promoters
• These promoter elements are found to be sites of
  interaction with protein factors, such as sigma

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Consensus features of prokaryote promoters

• -35 box - TTGACA
• -10 box - TATAAT
• Start point of transcription, usually a purine,
  often in the sequence CAT
• Distance between these three elements distance
  is important, sequence is not

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-35 Box

• Centred 35 bases upstream of startpoint of
  transcription
• Has consensus sequence
• T82 T84 G78 A85 C54 A45
• Where T82 means that this position is T in 82 of
  promoters used to generate the consensus
• Thus sequences can vary considerably from
  consensus and still function

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-10 Box

• T80 A95 T45 A60 A50 T96
• Note AT rich nature of -10 box
• Required for strand separation by RNA polymerase
  during formation of open complex
• Suggests that RNA Polymerase interactions are
  most important with bases 1, 2 and 6

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-35 to -10 distance

• Is in the range 16 to 18 basepairs for most
  promoters
• In some promoters may be 15, 19 or 20 bases
• Determines the orientation of the -35 and -10
  sites with respect to the RNA polymerase and this
  affects the binding geometry
• Sequence between -35 and -10 boxes does not
  affect promoter efficiency, only distance

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Efficiency of a promoter depends on similarity to
consensus

• Promoters vary in the efficiency of transcription
• The promoters with sequences closest to the -35
  and -10 consensus are the most efficient
• By implication, the consensus is the best
  promoter sequence possible
• Most promoters have mismatches to the consensus,
  and these work at lower efficiency

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Effects of mutations on promoters

• Mutations which change the -35 and -10 sequences
  (or the distance between them) will affect
  promoter efficiency
• Mutations generally cause reduction in efficiency
  - down mutations
• But up mutations also possible
• If the mutation changes the sequence to be closer
  to the optimal consensus, then it should be an
  up mutation
• Mutations which change the -10 or -35 boxes to
  make them less like the consensus will be down
  mutations
• Down mutations are more likely to occur on a
  numerical basis

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-35 and -10 mutations have different effects

• Down mutations in the -35 box reduce the rate of
  formation of the closed complex (KB) because the
  -35 box is recognised by the RNA polymerase, and
  especially the sigma subunit
• Down mutations in the -10 box reduce the
  conversion of the closed complex to the open
  complex (k2) which is related to the efficiency
  of melting the DNA helix

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Spacing of consensus sequences affects efficiency
of promoters

• Optimal distance for -35 to -10 box is 17 bp
• Increasing or decreasing that distance will make
  the promoter less efficient
• This is due to the orientation of the -35 and
  -10 boxes being changed as the length between
  them changes, as they are rotated around the DNA
  helix

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RNA Pol interactions with DNA

• Mapping protein-DNA interactions at a promoter
  allows characterisation of RNA Pol binding site
• Experiment involves using DNA Footprinting
  techniques
• Characterises exposure of DNA to enzymes or
  chemicals
• Protected areas are due to protein binding

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DNA footprinting shows interaction sites of
promoters with RNA Pol

• Cloned promoter fragment is labelled at one end
  (5 or 3)
• RNA Polymerase is bound to DNA
• Partial digestion undertaken with DNAase I to
  give one cleavage per DNA molecule
• Fragments resolved on gel

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DNA fragments show binding site for RNA
Polymerase on promoter
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Fragments resolved by gel electrophoresis to show
footprint
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Contacts sites can be mapped with a range of data

• Protection from DNAase I digestion
• Protection from chemical modification of C and G
  residues by RNA Pol binding
• Chemical modification results in blocking RNA Pol
  binding to DNA
• Analysis of effects of mutations in promoter
  sequence on RNA Pol binding (up and down
  mutations)

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RNA Pol binds to one side of DNA helix in the
promoter sequence
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DNA in unwound in RNA Pol open complex

• Can use chemical reactivity to map single
  stranded DNA in RNA Pol open complexes
• Can show that the promoter region is unwound from
  bases -9 to 3
• Results in supercoiling of DNA

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Transcription results in supercoiling of DNA
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Effects of supercoiling

• DNA must be unwound by RNA pol to introduce
  single stranded template region
• Results in overwinding before RNA Pol, and
  underwinding behind
• Supercoiling must be relaxed by enzymes
• Topoisomerase I relaxes -ve supercoils
• Gyrase introduces -ve supercoils
• Both are required for RNA synthesis to proceed
  efficiently

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Sigma factors control groups of genes

• Promoter sequence selectivity is controlled by
  sigma factor interactions with consensus
  sequences
• Different sigma factors are found, each one with
  a different sequence specificity
• These can therefore control groups of genes with
  related functions
• All sigma factors use the same RNA Pol core enzyme

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s70 controls general transcription

• The majority of promoters use s70
• s70 levels are not induced
• Basal efficiency of promoters controlled by
  consensus sequences interacting with s70
• Regulated level of transcription controlled by
  repressor and activator proteins

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?70 is a multifunctional, sequence specific DNA
binding protein
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N terminal domain of s70 blocks DNA binding

• The N terminal domain of s70 folds back to
  prevent s70 from binding to the promoter sequence
  in the absence of the RNA Pol core enzyme
• When part of the Holoenzyme, the N terminal
  domain probably bound to the core enzyme subunits
  to reveal DNA binding site
• If N terminal domain is deleted then s70 can bind
  to DNA in sequence specific fashion at promoters?

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Structural determinants of s70 specificity of
binding to -10 region

• Figure 11.21
• Interaction of s70 with DNA is through a-helices
  binding into major groove of DNA
• Can map this to regions conserved in other s
  factors
• Region 2 interacts with -10 consensus
• Region 4 interacts with -35 consensus
• Side chains on a helices determine DNA sequence
  specificity

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Other s factors control specific classes of gene
expression
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Sigma factors have different consensus structures
in promoters
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Transcription patterns in Bacillus species are
controlled by s factors

• Bacillus species have more than 10 s factors
• Control pathways the same as for E.coli
• But also control phage gene expression
• Also control sporulation (which does not occur in
  E.coli)
• Provides method of controlling gene expression in
  time dependent sequences

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Phage SPOI lifecycle in B.subtilis

• Early genes transcription controlled by sA
• Gene 28 encodes s factor for middle gene
  expression, replacing sA
• Genes 33 and 34 encode proteins that replace
  gp28 and control late expression
• Overall life cycle controlled in time by s factor
  expression

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Bacillus sporulation

• Under conditions on starvation or other
  environmental stress Bacillus species undergo
  sporulation pathway
• Generates highly resistant spores that can last
  for long periods in a dormant phase, resistant to
  wide range of environmental conditions
• Return to favourable conditions results in
  germination and growth
• Very important because of disease causing Bacilli
  (eg anthrax)

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Bacillus sporulation - s factors

• Figure 11.24
• Complex interplay between Mother cell and
  forespore with the formation of a septum and
  expression of two sets of s genes
• Allows for the expression of a sequence of genes
  resulting in the assembly of the spore structures
  and the time controlled sequence of expression of
  required gene products

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Termination of transcription

• Termination of transcription occurs at specific
  sequences at the 3 of the gene/operon
• Termination can be rho-independent or
  rho-dependent (rho is a protein termination
  factor)
• Most termination at the 3 end of operons is
  rho-independent

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Rho independent (intrinsic) termination

• Rho-independent termination sites are hairpin
  loop structures
• GC stem and U4-6 are required
• Formation of hairpin loop causes RNA Pol to pause
• AU run allows dissociation of RNA from DNA

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Rho (r? dependent termination

• 46kD monomer subunit, 275kD hexamer
• RNA dependent ATPase
• Requires RNA of gt50 bases long
• Binds to RNA and migrates in the 3 direction in
  an ATP utilising reaction
• Has as 5 -3 helicase that can separate RNA from
  DNA

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Rho binds to specific sequences

• Rho binds to C rich sequences in RNA upstream of
  termination site
• Rho dependent termination is relatively rare
• But rho is an essential protein in E.coli

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Mechanism of action of Rho

• Rho binds to site on free RNA in transcription
  complex
• Migrates towards RNA Pol using ATP
• Pause in transcription allows rho to catch up
• RNADNA hybrid is unwound and RNA is released

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Coupling of transcription and translation

• For rho to function it must be able to catch up
  with RNA Pol
• Usually ribosomes block this
• Tight coupling of transcription and translation
  prevent rho from prematurely terminating
  transcription
• But loss of translation (for example with a
  premature termination mutant) will allow rho to
  terminate transcription for the operon
• This may result in the loss of transcription of
  genes downstream in the same operon (polarity)

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Polarity in termination mutants
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Anti-termination as a mechanism for the
regulation of gene expression

• Anti-termination provides a mechanism for read
  through between transcription units
• Used particularly by bacteriophages to provide a
  timing mechanism for early/late switch of gene
  expression

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Anti-termination mechanism
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Summary

• RNA Polymerase transcribes mRNA, rRNA and tRNA in
  prokaryotes
• Regulation of transcription is at initiation
• Promoter selection is through sigma factors
• Efficiency of promoters is due to consensus
  sequences at -35 and -10
• Termination is at specific sites, and is
  generally constitutive, but can sometimes be
  regulated