Prokaryotic transcription Flashcards
central dogma of biology
DNA is transcribed to RNA which is translated into proteins.
Only possible reversal in direction occurs from retroviral ability to reverse transcribe RNA to DNA
advantages of having mRNA as a mediator molecule in transcription
- allows cell to separate storage of info from utilisation of info (safe storage in nucleus)
- greater amplification of synthetic output (1DNA molecule codes for multiple mRNA molecules)
mono vs polycistronic mRNA
monocistronic: each mRNA molecule produces one type of protein
polycistronic: each mRNA molecule contains info that can produce multiple discreet proteins
advantages of polycistronic mRNAs (3)
- usually proteins produced share a related function, so polycistronic mRNAs can ensure an almost equimolar amount of each protein in the cell
- saves energy (reduces unnecessary transcription)
- decreases required size of genome
promoter definition
Region of nucleotides within DNA that allows binding of certain proteins (eg, RNAP)
RNAP enzyme forms (2)
- CORE ENZYME: 2 alpha units, beta, beta prime and omega unit. High affinity for DNA, low for promoter, hence is used in elongation
- HOLOENZYME: core enzyme + sigma subunit. High affinity to promoter, low to DNA hence used for formation of initiation complex
HICE: holo initiation, core elongation
Structure of a prokaryotic promoter
-35 region: responsible for binding of sigma factor hence closed complex formation
-10 region (pribnow box): responsible for open complex formation
+1 region: start of coding sequence
Role of 3’ and 5’ UTRs
Untranslated regions that are between promoter and coding sequence (5’) or between coding sequence and terminator (3’)
protect the mRNA from being degraded
up/down elements
can be present in the sequence between -35 and -10 of promoter and either upregulate or downregulate gene transcription
Promoter strength def
number of transcripts that are initiated by a promoter per unit time.
Factors affecting promoter strength (4)
- affinity of RNAP to promoter (regulated by -35)
- efficacy of closed complex to open complex conversion
- efficacy of promoter clearance
- presence of an up element (increases strength)
Basal transcription def
average rate of transcription in the absence of an activator or repressor element
Structure and function of activators
Activators are TFs that bind to DNA increasing the strength of a promoter
Increase time that RNAP can bind with operator without dissociating so it helps the initiation of closed complex
Structure and function of a repressor
TF that have binding sequences overlapping with the promoter
Binds to operator to prevent RNAP from binding there itself –> no transcription if present
Factors affecting extent of activation/repression
- conc of element present
- affinity of element to DNA
Initiation process
-creation of holoenzyme from binding of sigma subunit and free cytoplasmic core enzyme
-collision of holoenzyme with DNA and sliding along strand until it recognises the promoter region
-closed complex forms when RNAP binds to promoter
-RNAP denatures double helix and forms an open complex
-formation of transcription bubble and addition of a small number of rNTPs
-sigma factor is discarded and RNAP breaks away from promoter to enter elongation phase
Elongation process
-dissociation of sigma factor causes conformational change into claw shape which increases enzyme’s synthesis speed
-transcription bubble is constantly expanding downstream and contracting at the enzyme’s rear
-reaches termination signal
-loss of affinity to DNA and so enzyme disengages
Termination process
caused by reaching termination signal
2TYPES:
1. RHO INDEPENDENT:
-termination signal is GC rich palindrome followed by a long polyA
-structure of the palindrome sequence during transcription is a hairpin loop
-causes DNA/RNA hybrid to pause
-A rich region causes release of transcript and RNAP
- RHO DEPENDENT:
-requires Rho termination factor (hexameric protein)
-GC palindrome sequence is present but no poly A
- Rho factor binds to RUTS (rho utilization site)
-the pause of RNAP caused by hairpin loop allows Rho to move into termination region and unwind DNA-RNA complex
operon def
An operon is a cluster of genes under a single promoter, hence controlled together
Positive and negative transcription control
POSITIVE: occurs usually for weak promoters with a low affinity for RNAP: basal transcription is low or non (due to weakness) and so an activator binds to activator binding site to increase transcription rate
NEGATIVE: occurs usually for strong promoters with high affinity for RNAP: basal transcription is high and so repressors bind to operator to lower/stop transcription rate
LAC OPERON structure
order of upstream-downstream:
-LacI repressor site
-CAP (activator binding) site
-promoter
-operator
-LacZ: beta galactosidase
-LacY: lactose permease
-LacA
2 factors that affect transcription rate of lac operon in different environments
- ALLOLACTOSE conc: binds to repressor and can dissociate it from operator
- cAMP and CAP complex conc: binds to CAP site and increases rate of transcription
what is the relationship between levels of glucose and levels of cAMP?
inverse proportion,
high glucose levels = low cAMP
low glucose levels = high cAMP
lac operon: no lactose no glucose
NO TRANSCIRPTION:
1. no allolactose present so the receptor is transcribed, translated and bound to operator
2. high levels of cAMP so the CAP-cAMP complex is formed but this doesn’t change the fact that RNAP cant bind to promoter
lac operon: lactose present without glucose
HIGH TRANSCIRPTION RATE
1. allolactose present which allows the receptor to bind to it and dissociate from operator (RNAP binds to promoter)
- cAMP levels are high so high conc of CAP-cAMP complex, which increases trasncirption
lac operon: both lactose and glucose
LOW TRANSCRIPTION RATE:
1. allolactose present which allows the receptor to bind to it and dissociate from operator (RNAP binds to promoter)
- cAMP in low levels so no cAMP-CAP complexes formed meaning no increase in transcription rate
lac operon: glucose present without lactose
NO TRANSCIRPTION:
1. no allolactose present so the receptor is transcribed, translated and bound to operator
2. low levels of cAMP so the CAP-cAMP complex is not formed
cis vs trans acting elements
cis acting: short DNA stretches of a defined sequence that act as binding sites
trans acting: proteins (eg. TFs/repressors) that bind to cis acting factors
possible mutations of the lac operon (2)
- Lac1- mutant: no functional repressor is present (RECESSIVE)
- Lac0- mutant: no functional operator that the repressor can bind to (DOMINANT)
