Prokaryotic transcription

Question 1

central dogma of biology

Answer 1

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

Question 2

advantages of having mRNA as a mediator molecule in transcription

Answer 2

1. allows cell to separate storage of info from utilisation of info (safe storage in nucleus)
2. greater amplification of synthetic output (1DNA molecule codes for multiple mRNA molecules)

Question 3

mono vs polycistronic mRNA

Answer 3

monocistronic: each mRNA molecule produces one type of protein

polycistronic: each mRNA molecule contains info that can produce multiple discreet proteins

Question 4

advantages of polycistronic mRNAs (3)

Answer 4

1. usually proteins produced share a related function, so polycistronic mRNAs can ensure an almost equimolar amount of each protein in the cell
2. saves energy (reduces unnecessary transcription)
3. decreases required size of genome

Question 5

promoter definition

Answer 5

Region of nucleotides within DNA that allows binding of certain proteins (eg, RNAP)

Question 6

RNAP enzyme forms (2)

Answer 6

1. CORE ENZYME: 2 alpha units, beta, beta prime and omega unit. High affinity for DNA, low for promoter, hence is used in elongation
2. HOLOENZYME: core enzyme + sigma subunit. High affinity to promoter, low to DNA hence used for formation of initiation complex

HICE: holo initiation, core elongation

Question 7

Structure of a prokaryotic promoter

Answer 7

-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

Question 8

Role of 3’ and 5’ UTRs

Answer 8

Untranslated regions that are between promoter and coding sequence (5’) or between coding sequence and terminator (3’)

protect the mRNA from being degraded

Question 9

up/down elements

Answer 9

can be present in the sequence between -35 and -10 of promoter and either upregulate or downregulate gene transcription

Question 10

Promoter strength def

Answer 10

number of transcripts that are initiated by a promoter per unit time.

Question 11

Factors affecting promoter strength (4)

Answer 11

1. affinity of RNAP to promoter (regulated by -35)
2. efficacy of closed complex to open complex conversion
3. efficacy of promoter clearance
4. presence of an up element (increases strength)

Question 12

Basal transcription def

Answer 12

average rate of transcription in the absence of an activator or repressor element

Question 13

Structure and function of activators

Answer 13

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

Question 14

Structure and function of a repressor

Answer 14

TF that have binding sequences overlapping with the promoter
Binds to operator to prevent RNAP from binding there itself –> no transcription if present

Question 15

Factors affecting extent of activation/repression

Answer 15

1. conc of element present
2. affinity of element to DNA

Question 16

Initiation process

Answer 16

-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

Question 17

Elongation process

Answer 17

-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

Question 18

Termination process

Answer 18

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

2. 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

Question 19

operon def

Answer 19

An operon is a cluster of genes under a single promoter, hence controlled together

Question 20

Positive and negative transcription control

Answer 20

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

Question 21

LAC OPERON structure

Answer 21

order of upstream-downstream:

-LacI repressor site
-CAP (activator binding) site
-promoter
-operator
-LacZ: beta galactosidase
-LacY: lactose permease
-LacA

Question 22

2 factors that affect transcription rate of lac operon in different environments

Answer 22

1. ALLOLACTOSE conc: binds to repressor and can dissociate it from operator
2. cAMP and CAP complex conc: binds to CAP site and increases rate of transcription

Question 23

what is the relationship between levels of glucose and levels of cAMP?

Answer 23

inverse proportion,
high glucose levels = low cAMP
low glucose levels = high cAMP

Question 24

lac operon: no lactose no glucose

Answer 24

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

Question 25

lac operon: lactose present without glucose

Answer 25

HIGH TRANSCIRPTION RATE
1. allolactose present which allows the receptor to bind to it and dissociate from operator (RNAP binds to promoter)

2. cAMP levels are high so high conc of CAP-cAMP complex, which increases trasncirption

Question 26

lac operon: both lactose and glucose

Answer 26

LOW TRANSCRIPTION RATE:
1. allolactose present which allows the receptor to bind to it and dissociate from operator (RNAP binds to promoter)

2. cAMP in low levels so no cAMP-CAP complexes formed meaning no increase in transcription rate

Question 27

lac operon: glucose present without lactose

Answer 27

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

Question 28

cis vs trans acting elements

Answer 28

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

Question 29

possible mutations of the lac operon (2)

Answer 29

1. Lac1- mutant: no functional repressor is present (RECESSIVE)
2. Lac0- mutant: no functional operator that the repressor can bind to (DOMINANT)