Zusammenfassung
Summary Molecular Microbiology: both partims (VUB)
- Kurs
- Hochschule
Summary of all lectures of the course Molecular Microbiology, given by professors Remaut, professor Peeters / dr. Bervoets at the VUB.
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Inhaltsvorschau
MOLECULAR MICROBIOLOGYREGULATION MECHANISMS
TOPIC 1: REGULATION OF TRANSCRIPTION – TRANSCRIPTION FACTORS
LEVELS OF REGULATION
• Microorganisms (MO’s) react to various environmental conditions
→ sensing → responding by regulation
→ Nutritional conditions, stress conditions, cell-cell interactions
→ Different modes of regulation in response:
▪ Transcriptional
▪ Translational
▪ Metabolic
• Picture: a bacterial system with a typical bacterial promotor (not in
archaea)
• Prokaryotes: a coupled transcription & translation ↔ not in eukaryotes: cell compartments
TRANSCRIPTION
• Euk: RNA polymerase II
• The archaeal basal transcription machinery is a simplified version of the
eukaryotic RNA polymerase II machinery + basal transcription factors =
homologues of euk TF’s
• Bacteria: RNA polymerase with typical structure as in eukaryotes
RNA POLYMERASES
➔ Bacterial RNAP is the basis for euk/arch RNAP
TRANSCRIPTION IN BACTERIA
• Bacterial promotor
→ -35 sequence: TTGACA
→ -10 sequence (Pribnow box): TATAAT
→ Always 16-18 bp spacing in between them
• Bacterial RNA polymerase
→ Single RNAP (one type)
→ Core enzyme
▪ Catalyzes RNA synthesis
▪ 5 subunits: two α subunits, β, β’ (prime), ω
→ Holoenzyme
▪ If the core enzyme bound to an associated σ (sigma) factor (orange)
▪ The σ factor (most species have multiple σ factors) assists the core
enzyme by recognizing the promotor → mediates reaction with DNA
• Recognition of -35 and -10 elements
1
, • When RNAP binds the promotor (in -35 to -10
region (purine rich)), they form a closed complex:
→ RNAP unwinds the DNA (helicase activity)
→ Forming an open complex
→ A region of 16-20 bp unwound DNA
becomes the transcription bubble which
moves with the polymerase
→ The sigma factor dissociates from the core enzyme after
initiation of transcription → available for other RNAP’s
• Multiple RNAP’s bind at the same time on DNA
→ Multiple transcripts at the same time
• Termination of transcription:
→ A stem-loop in RNA (secondary structure) = signal to
end the transcription
• Result: poly-cistronic mRNA
→ DNA < multiple genes → mRNA codes for multiple proteins
TRANSCRIPTION IN ARCHAEA
• The archaeal promotor resembles the eukaryotic promotor
→ BRE element: purine rich factor B recognition element (-33)
→ TATA box (-25)
→ Initiator region
→ Downstream promotor element (only eukaryotes!)
• TBP (TATA binding protein) recognizes TATA box
• TFB (transcription factor B) binds the TBP-DNA complex and determines the orientation
of transcription by recognizing BRE
→ TFB = an asymmetrical protein:
▪ A part recognizes the complex
▪ Another part recognizes BRE and bind in major groove of BRE
• RNAP binds the TFB-TBP-DNA complex (= pre-initiation complex PIC) and transcription is
initiated
• Some archaea have multiple TBPs and TFBs (cfr. sigma factors in bacteria?)
TRANSCRIPTION REGULATION BY TRANSCRIPTION FACTORS
• Regulation of transcription initiation occurs by the action of regulatory proteins = transcription factors:
→ Sense signals (ligand interaction (= direct ; metals, metabolites, toxic compounds…), posttranslational
modifications (= indirect ; often via phosphorylation)…)
→ Bind on TF binding sites (TFBSs) in promotor region on the DNA
→ Activate or repress transcription initiation by interacting with basal transcription machinery
▪ Eg. MO senses glucose in envir → activate genes encoding enzymes that use glucose → °E
→ Operon = group of genes on same location regulated by same TF
→ Regulon = group of genes regulated by the same TF
2
,TRANSCRIPTION FACTOR STRUCTURE
Classes based on domain architecture of prokaryotic TFs
• Standalone copies of a DNA-binding domain (eg. ci repressor, Fis)
→ Very small ; only harbor a dna binding domain
→ Often via phosphorylation (posttr. mod)
• Single-component system: one protein, usually < 2 domains: → we will focuss on this in this chapter!
→ DNA-binding domain
→ A stimulus sensor module: directly interact with small-molecules ligand → °allosteric response
• Two-component system (consists of two separate proteins) → next chapter
→ Signal transduction by phosphorylation
→ Membrane bound: sensing extracellular signals
→ Also a DNA-binding domain
→ Prevalent in bacteria
OCCURRENCE OF TRANSCRIPTION FACTORS
• The number of single-component TFs in bacterial/archaeal genomes
scale nonlinearity with proteome size
• Complex lifestyles require a higher proportion of TFs and transcription
units to better orchestrate a response to changing conditions →
different lifestyles
• No fluctuations in envir: eg. intracellular pathogens or parasitic MO’s
→ dependent on conditions of host → not a large genome and TFs
TRANSCRIPTION FACTOR STRUCTURE: FURTHER
Prokaryotic TFs have a two-domain structure:
1) (winged) helix-turn-helix DNA-binding domain < α-helices
→ Recognition α-helix interacts with major groove of the DNA
▪ Will determine which sequence is recognized by TF!
→ Stabilizing α-helix positions recognition helix
→ Sometimes a third α-helix at the stabilizing α-helix
2) Ligand-interaction domain
→ Ligands (also called effectors or co-factors) can vary widely in size and nature
(small ions, nucleotides, sugars, peptides…)
They form homodimers or higher oligomers of dimers < two TFs together!
• Protein-protein contact domain hold both monomers together
• 2x a DNA-binding domain → mirror images (see further)
TF families
• Bacteria and archaea have the same TF families: TetR, Lrp, LTTR, CopG, GntR…
→ Although their basal transcription mechanisms are different
• Classification based on ligand binding domain → so which ligand they bind
• Families are structurally, not functionally, defined → members can have widely varying functions
• Family names = based on first discovered TF within that family
→ Eg. TetR: repressor for E. coli that interact with tetracycline (an AB) ; but other members can have
completely different function (eg. acetyl co-A as ligand)
• Helix-turn-helix = most common DNA binding motif
3
, • Scheme: EBD (effector (= ligand) binding domain)
has a complex hexameric structure → so not always
dimeric! → can bind with multiple DNA’s at once ;
eg. Arginine as ligand
• DBD (dna binding domain) is always very similar
• EBD has more structural variation (ligands) +
responsible for dimerization!
• Eg. FadR → ligand = fatty acid acetyl co A
• Eg. TetR → regulator for E.coli
MECHANISMS OF DNA BINDING
TFBSs
• Length between 12 and 30 bp
→ If small dimeric TFs → recognize 1 major groove → 12 bp
→ If large dimeric TFs → recognize 3 adjacent major grooves → up to 30 bp
• Allosteric effect in TF:
→ Distance between the two helices = crucial → needs to be optimized so both monomers can optimally
dock into the major grooves
→ If distance too large/small → no DNA-binding
→ Structural conformation change as the ligand binds the TF →
allostericity is passed on to DNA-binding sites → regulation of the
distance
• Inverted repeats (palindromic sites) that reflect the homodimeric nature of
the TFs
→ The second one recognizes an inverted repeat of the sequence
recognized by the first one
• Consensus sequence represents variability of the motif at different targets
→ Most TFs bind multiple sites in genome → those genes have almost
identical recognition sequence
CALCULATING THE CONSENSUS SEQUENCE
• Graph A: all genes that can be bound be a certain TF with their seq.
• Consensus sequence = sequence of preferred nucleotides
→ By counting number of occurrences → take most common
• Position weight matrix: weighed frequencies of the occurrence of
nucleotides at specific positions
• Sequence logo: graphical representation of PWM: ordered stack of letters in
which the letter’s height indicated the amount of information at that
position
• This is a very good method!
REGULATION MECHANISMS OF TRANSCRIPTON FACTORS
• Multiple outcomes after DNA-binding are possible:
1) The binding event can block/repress transcription = negative regulation by TF
2) The binding event can activate transcription = positive regulation by TF
• This depends on the ligand! → a single TF can have multiple outcomes
4
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