prokaryotic transcription

Explain nucleotide polymerization differences to DNA synthesis

• RNA polymerase uses DNA template (non-coding/antisense (-) strand) => DNA-directed (or DNA-dependent) RNA polymerase

• Polymerizes ribonucleotides (NTPs) not deoxyribonucleotides (dNTPs)

• Uracil instead of Thymine

- Coding strand runs 5' to 3'. Non coding strand runs 3' to 5'

Whatre the general concepts of prokaryotic transcription?

• RNA needs to be synthesized to carry message for protein synthesis to ribosome

• Transcription can only occur if there is a template

• Non-coding DNA strand serves as template for transcription

• Enzymes facilitate transcription

• Synthesis happens from 5’ to 3’ (incoming nucleotides incorporated at 3’ hydroxyl end)

• RNA molecules less stable than DNA => rapid turnover

Whats the general overview of prokaryotic transcription

• Region of dsDNA dissociated

• RNA polymerase binds to promoter region

• RNA polymerase polymerize ribonucleotides into RNA strand

• In prokaryotes, usually only a single RNA polymerase

• In prokaryotes, transcription is coupled with translation

Whatre the important proteins for prokaryotic transcription?

• RNA Polymerase – Enzyme that polymerizes NTPs using a DNA template

• Sigma (σ) factors – Specificity factors that recognizes key sequence motifs for determining which genes to  transcribe

Whatre the important parts of the open reading in prokaryotes?

• In DNA its -35 and -10 which are the RNA polymerase recognition site and RNA polymerase binding box (pribnow box) which are both on the promoter

• In mRNA its the shine Dalgarno sequence and translation start (initiation codon)

Whatre the 2 parts of the RNA polymerase holoenzyme and what subunits are in them?

Core enzyme (α, α, β, β’ and ω subunits)
• Catalytic activity of the RNA pol
• Polymerize ribonucleotides

Holoenzyme
• Core enzyme + sigma σ factor
• σ factor is a multi-subunit protein
• σ factor has no catalytic activity
• σ factor recognize the start of a gene

Whatre the important features of initiation of transcription and order of events?

• Sigma recognizes -35 element

• Entire holoenzyme binds to promoter at -10 element

• Unwinds DNA to form open complex which causes....
• Transcription bubble
• ~ 16 – 20 bp inside open complex
• sigma factor dissociates

• Core enzyme responsible for elongation of transcription

• -35 and -10 are consensus sequences
• Both are located on the coding strand

• Variations determine how easily RNA pol will recognize and bind the promoter
• This affects the efficiency of initiation

Explain the steps of what happens in transcription initiation in prokaryotes

• Binding of RNA polymerase holoenzyme to form a closed complex where the DNA is still double stranded

• An open complex then forms where the DNA is unwound

• The sigma factor is released after open complex is formed and the RNA polymerase core enzyme just keeps moving on

Explain elongation in transcription for prokaryotes

• Core enzyme reads the template strand of DNA (3′ to 5′)
  • Synthesizes RNA (5′ to 3′)
  • Adds rNTPs
  • Makes polymer of rNMPs as Pi is released to create energy used to add phosphate bonds of RNA itself

• Temporary dsRNA/DNA hybrid is formed

• RNA pol moves downstream and dsDNA re-anneals behind it

• Causes the RNA strand to melt away from the DNA template

Explain termination of transcription and look at steps in notes

• Transcription terminator
  • Is an inverted repeat with a central non-repeating unit
  • When transcribed it forms a hairpin in the RNA
  • Causes RNA pol to temporarily stall

• Releases core enzyme to re-associate with sigma factor/s

Whatre the 2 ways RNA pol can be removed from DNA?

Rho independent termination

Rho dependent termination

Explain Rho independent termination

• RNA pol transcribes the inverted repeats
  • RNA forms a hairpin
  • Causes RNA pol to pause while transcribing an A-rich region of the template
  • Produces string of 6Us in RNA following the hairpin (poly-U)

• DNA and RNA separate

• RNA pol releases the DNA

• Intrinsic terminator

• no special protein needed

• More common than Rho dependent as it doesnt need energy or permission from promoter

Explain Rho dependent termination and look at steps in notes

• Requires a protein
  • Which is  rho (ρ) factor which is a multi-domain protein with an ATPase domain and rut site on mRNA
  • Moves along mRNA until it reaches halted RNA pol at hairpin
  • Removes RNA pol at hairpin
  • May also be responsible for tightly paired RNA-DNA hybrid molecules (often at operons)

Explain the post transcriptional modifications

• Some bases in RNA are modified after transcription

• Unusual bases in tRNA such as...
  • Dihydrouridine(D) ->  fully saturated pyrimidine ring (no double bonds)
  • Pseudouridine (ψ) -> ribose joined to C5 instead of N1
  • Ribothymidine (T) -> Methyl group is added to C5 of uridine

• Limited other post-transcriptional processes in prokaryotes

Explain operons in prokaryotes and what they are

• Gene clusters on the genome where all the genes are co-regulated

• Typically, genes in an operon have related function/s

• Genes required under the same condition may be coupled in their transcription

• All genes in operon transcribed into a single mRNA molecule which are called (polycistronic mRNA → one promoter regulates bunch of things differently)

-Prokaryotes usually have more polycistronic genes as opposed to eukaryotes, as it can do more things at once, as there’s less regulation and things compared to prokaryotes

Explain the general idea of transcriptional regulation

• If all genes were actively transcribed all the time, it would be energetically highly inefficient → to produce mRNAs encoding proteins that are not necessary at that point in time is expensive

• Promoters are not transcribed BUT are key to assembling a transcription complex
  • Controls when/how much a gene is transcribed

Whatre the 2 main methods of transcriptional regulation?

• By promoter strength

• DNA binding proteins

Whats the difference when the affinity for the RNA pol in strong promoter vs weak promoter?

• Strong promoter
  • RNA pol has high affinity for promoter
  • More efficient transcription
  • More mRNA produced = more protein

• Weak promoter
  • RNA pol has low affinity for promoter
  • Less efficient transcription
  • Less mRNA produced = less protein

Explain transcriptional regulation of DNA binding proteins

• Genes (via their products) control other genes
  • Require cis and trans elements
  • Can respond to environmental factors
  • Via effectors molecules

• Use positive and negative regulation mechanisms

Explain negative regulation of DNA binding proteins and the difference in effectors

•  Upstream of promoter

• DNA binding proteins (repressors) to physically block RNA pol from transcribing the operon
  • Repressor binds at operator site downstream of promoter
  • Blocks RNA pol from getting through

• Effectors:
• Inducers - prevent the repressor protein from binding operator (prevents it from blocking RNA pol)
• Co-repressors - Help repressor protein bind operator DNA (blocking of RNA pol)

Explain positive regulation of DNA binding proteins and the difference in effectors

• Downstream of promoter

• DNA binding proteins (activators) to physically help the RNA pol find the promoter and thus transcribe the operon
  • Activators bind the activator binding site (ABS) located upstream of the promoter
  • Helps recruit RNA pol to the promoter

• Effectors:
• Inducers - Binds the activator protein and allows it to bind ABS (help recruit RNA pol to the promoter)
• Repressors - Binds the activator protein and prevents it from binding ABS (No longer can help bring RNA pol to the promoter)

Whatre the 2 types of promoters?

Inducible and repressible promoters

Explain inducible promoters

• Need to be turned on

• Default state of promoter is off -> induced to switch transcription on

• Negative regulation = repressor is bound, and inducer/s need to bind to remove repressor from promoter region

• Positive regulation = promoter not recognized effectively unless an activator protein recruits RNA Pol

Explain repressible promoters

• Default state is on -> repressed to switch transcription off

• Negative regulation = co-repressor needs to activate repressor for it to bind to promoter region

• Positive regulation = repressor inactivates activator protein to prevent recruitment of RNA Pol

Whats the general difference between inducible and repressible operons?

• Inducible operons are “typically” switched off -> need to be switch on when appropriate (induced)

• Repressible operons are “typically” switched on -> need to be switched off when appropriate (repressed)

Explain the features/ general idea behind transcription regulation of housekeeping genes

• Some related housekeeping genes are scattered across the genome, but need to be expressed at the same time

• Some related housekeeping genes are clustered but individually expressed

• Some related housekeeping genes are clustered and co-regulated

• Actual architecture might be organism dependent, e.g., gyrase genes

Explain the features of constitutively expressed operons

• Operons encoding proteins associated with housekeeping functions (replication, transcription, translation etc.)

• Doesn’t require complex regulation, as expression of genes is constantly required e.g.,
  • Ribosomal operon/s
  • RNA Pol operon/s
  • DNA Pol operon/s

Explain negative regulation when default state of operon has transcription switched off

• Negative regulation  = Always talking about repressor
→Repressor preventing transcription of the operon (in native form)
→Inducer bound to repressor to inactivate repressor
→Expression turned on
→When repressor attached, inducible operon turned off

Explain positive regulation when default state of operon has transcription switched off

• Positive regulation = needs an inducer
→Activator protein not bound to ABS to actively recruit RNA Pol initially
→Inducer binds to activator which allows binding to ABS and active RNA Pol recruitment

Whatre the main points of an operon?

• Gene clusters on the genome where all the genes are co-regulated

• Typically, genes in an operon have related function/s

• Genes required under the same condition may be coupled in their transcription

• All genes in operon transcribed into a single mRNA molecule (polycistronic mRNA)

Whats the Ara operon and what does it do?

• Operon that encodes all proteins required metabolizing L-arabinose to D-xylulose- phosphate

• Its an inducible operon

How does ara operon start off?

• AraC protein is bound to operator region to prevent transcription of PC and PBAD which are both promoters that run in the opposite direction and are involved in the metabolism of arabinose

• AraC protein is bound to operator region to prevent transcription of PC and PBAD

• The homodimer AraC contains an arabinose-binding pocket

What happens to ara operon in presence and absence of arabinose

• In the presence of arabinose, the AraC homodimer functions as inducer with arabinose bound and allows expression of PC and PBAD, but also of PE and PFGH that are associated with arabinose uptake

• In the absence of arabinose, the ara operon is repressed because the regulatory protein AraC acts as a repressor. Without arabinose bound to it, the AraC dimer binds simultaneously to the distant araO₂ operator and the araI₁ half-site. This dual binding forces the intervening DNA to bend into a DNA loop, which physically blocks RNA polymerase from binding to the P_BAD promoter and prevents the transcription of the metabolic genes (araB, araA, and araD)

LOOK AT STEPS

Whats the Trp operon?

• Operon that encodes all proteins required for tryptophan biosynthesis

• Trp operon switched off when Trp levels are high

• Trp is essential for producing proteins.

Explain what happens when transcription of Trp operon can occur

• Trp biosynthetic pathway is switched on to produce Trp from chorismite

• Although TrpR repressor protein is present in the cytoplasm (constitutively expressed at low levels), it is present in an inactive form -> Transcription of the operon can occur

Explain what happens when transcription of Trp operon cannot occur

• When Trp is present in the cytoplasm, Trp acts as a corepressor
  • Trp binds to TrpR repressor and activates the repressor

• Activated repressor binds to operator region and prevents transcription by blocking RNA Pol

• No transcription of the operon can occur

Whatre the 3 important parts of the lac operon and what do they do?

• Β-galactosidase (Lac Z) - Breaks lactose into glucose and galactose, also converts some lactose into allolactose which works as an inducer

• Lac permease (Lac Y) - It uses a proton gradient to actively transport lactose and other β-galactosides from the outside environment into the interior of the cell

• Transacetylase (Lac A) - it transfers an acetyl group from acetyl-CoA onto thiogalactosides. By acetylating non-metabolizable galactosides, it helps the cell pump them back out to prevent toxic build-up.

What kind of promoter does the lac operon have and what works as the inducer?

The lac operon should be switched on when lactose is present -> inducible promoter

Allolactose necessary to inactivate repressor

What are the 2 requirements that need to be met for the lac oepron?

• When two requirements are to be met, it means that there are multiple elements to the control of expression
  • Still have the lac repressor that needs to be inactivated (sensor for whether there is lactose)
  • Need a way to detect whether there is glucose present in the cell

What works for catabolite repression in the lac operon?

• Activator binding site (CAP site)
• Catabolite activator protein (CAP) (or CRP: cyclic AMP receptor protein)
• Cyclic AMP (cAMP)

• CAP-cAMP acts as an inducer

What happens to the lac operon when glucose is absent in terms of catabolite repression?

• When glucose is absent, cAMP is high
  • cAMP binds to CAP, Forms CAP complex
  • CAP-cAMP binds to activator binding site
  • Allows binding of RNA pol
  • Transcription can proceed

What happens to the lac operon when glucose is present in terms of catabolite repression?

• When glucose is present, cAMP is low
  • cAMP does not bind to CAP, NO CAP complex
  • CAP is inactive
  • Can’t bind activator binding site
  • RNA pol can’t bind promoter
  • Transcription is turned off

How are cAMP and glucose related

• Extracellular glucose is taken up by PTS which is the phosphotransferase system which makes glucose 6 phosphate 

• When glucose is present, PTS is busy moving glucose into the cell so no adenylate cyclase is activated

• When glucose is absent, PTS component activates adenylate cyclase cuz theres no need to pass on phosphate so adenylate cyclase is activated which Produces cAMP

Which enzyme activates adenylate cyclase?

• Enz IIa

Know the summary of lac operon