Summary
Summary Molecular Microbiology: both partims (VUB)
- Course
- Institution
Summary of all lectures of the course Molecular Microbiology, given by professors Remaut, professor Peeters / dr. Bervoets at the VUB.
[Show more]
Seller
Follow
lunawillems1
Reviews received
Content preview
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
The benefits of buying summaries with Stuvia:
Guaranteed quality through customer reviews
Stuvia customers have reviewed more than 700,000 summaries. This how you know that you are buying the best documents.
Quick and easy check-out
You can quickly pay through credit card or Stuvia-credit for the summaries. There is no membership needed.
Focus on what matters
Your fellow students write the study notes themselves, which is why the documents are always reliable and up-to-date. This ensures you quickly get to the core!
Frequently asked questions
What do I get when I buy this document?
You get a PDF, available immediately after your purchase. The purchased document is accessible anytime, anywhere and indefinitely through your profile.
Satisfaction guarantee: how does it work?
Our satisfaction guarantee ensures that you always find a study document that suits you well. You fill out a form, and our customer service team takes care of the rest.
Who am I buying these notes from?
Stuvia is a marketplace, so you are not buying this document from us, but from seller lunawillems1. Stuvia facilitates payment to the seller.
Will I be stuck with a subscription?
No, you only buy these notes for $11.97. You're not tied to anything after your purchase.
Can Stuvia be trusted?
4.6 stars on Google & Trustpilot (+1000 reviews)
79373 documents were sold in the last 30 days
Founded in 2010, the go-to place to buy study notes for 14 years now