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BMSC320 > Prokaryotic Gene Expression > Flashcards

Prokaryotic Gene Expression Flashcards

1
Q

what does transcription require?

A

binding of RNA polymerase to produce RNA from a gene
- prok have sincle RNA pol enzyme (5+1 protein complex w/ 5 core proteins + 1 additional protein)
- euk pol is similar structure with completely different proteins

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2
Q

What are the activities of RNA pol

A

5’ to 3’ polymerase activity
helicase activity

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3
Q

what are the core proteins in RNA pol

A

β, β’, α (x2) and ω
- supply RNA polymerase activity
- when given the right conditions it will produce RNA from any nicked DNA

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4
Q

What are the functions for the core proteins in RNA pol?

A
  • ω (omega) units help with assembly
  • α (alpha) units are required for assemble and have DNA binding specificity
  • β and β’ are catalytic proteins for RNA polymerization
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5
Q

what is the 1 associated protein in RNA pol?

A

sigma (σ)
- adds specificity to the enzyme

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6
Q

what happens during Pol assembly?

A

occurs in cytoplasm, not on DNA
1. the two α units dimerize
2. α2 binds β while ω binds β’
3. α2β complex binds the β’ω complex

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7
Q

how is the additional protein added to the core RNA polymerase?

A
  • core polymerase binds to DNA indiscriminately
  • protein σ (sigma) finds a core enzyme bound to DNA and associates with it
  • the holoenzyme now has sequence specificity and associates with promoters
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8
Q

how does σ find promoters?

A

transcription factors
- add sequence specificity to RNA pol
- in E. coli, σ₇₀ is the transcription factor used for most genes

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9
Q

How does σ₇₀ work in E. coli?

A
  • contacts DNA at 2 location to set where RNA pol will initiate
  • the sequence locations are found 10 and 35 bases upstream of the +1 base (first nt of DNA that gets transcribed into RNA)
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10
Q

What effects promoter variability and strength?

A
  • there’s a base sequence that σ binds to readily, but not every promoter has a perfect sequence
    • best fit is called consensus sequence
    • we don’t want every promoter to be the consensus
      sequence because we don’t want every gene
      expressed at the maximum level
  • closer match= increased probability of RNA pol binding = gene expressed more often
  • highly expressed genes must have very optimal -10 and -35 regions
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11
Q

Explain alternative promoters

A

there are additional σ factors that are used to turn on specific genes, but they are only present in certain conditions such as stress, nitrogen or glucose starvation, and heat
- each factor has its own unique consensus site, not necessarily at the -10 and -35 regions
- having the same single σ factor control many genes is a way to coordinate expression of the genes

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12
Q

Explain alternative promoters

A

there are additional σ factors that are used to turn on specific genes, but they are only present in certain conditions such as stress, nitrogen or glucose starvation, and heat
- each factor has its own unique consensus site, not necessarily at the -10 and -35 regions
- having the same single σ factor control many genes is a way to coordinate expression of the genes

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13
Q

what are 2 additional binding sites?

A

UP element (~40-60): bound by the α subunit (not σ) and consensus sequence is As and Ts
Extended -10: T-G sequence at -14,-15

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14
Q

Why is a transcriptional bubble formed at the -10 sequence?

A
  • the high A-T content makes it easier to denature the DNA to form transcriptional bubble/open promoter complex
  • will keep a ~17bp bubble open
  • note that initial RNA synthesis is slow, single bases don’t bind strongly so the 1st base may drift away before the 2nd base can come in
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15
Q

what are the 2 possible fates of transcriptional bubbles?

A
  1. “promoter escape” - getting past the 10 base RNA stage (supposed to happen because the first 10 bp are fragile)
  2. “abortive initiation” - RNA detaches before promoter escape occurs (can’t extend beyond 10 bp)
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16
Q

What happens to Sigma factor?

A
  • RNA and DNA must be able to feed through pol complex
  • exit channel is initially blocked by σ
  • for mRNA to be synthesized longer than 10 nt it must push σ out of the exit channel
  • usually σ is completely removed, sometimes remains attached
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17
Q

What is the error rate of RNA pol?

A

1/10^5 (100x lower than DNA)
- not a huge problem because mRNA will eventually break down

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18
Q

What are RNA pol’s 2 types of proofreading activity?

A
  1. Pyrophoshorolytic removal: reversal of ribonucleotide addition reaction (a backwards reaction using PPi and producing A/GTP)
  2. Hydrolytic removal: RNA pol backtracks about 5 bases and cuts by exonuclease activity (doesn’t really improve error rate without specificity for error). Enhanced by GreA and GreB elongation factors, will synthesize about 50 bases/second and pause every 100 bases to backtrack and fix errors
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19
Q

What are the two types of bacterial RNA termination?

A
  1. Rho independent
  2. Rho dependent
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20
Q

Describe Rho independent termination

A
  • utilizes GC-rich inverted repeat sequence, has no effect on DNA but causes stem-loop to form on RNA
  • When stem loop forms it pulls RNA from pol
  • needs a stretch of approx. 3+ A:U pairs so binding will be weak enough to free RNA
  • need a stop codon in mRNA so that ribosome will release it and allow it to form the stem loop
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21
Q

Describe Rho dependent termination

A
  • used less often than Rho independent
  • Rho is hexamer RNA binding protein that binds to RNA sequence called rut site (Rho UTilization)
  • requires naked RNA (region unbound by ribosomes) to bind
  • Rho binds mRNA and pulls it out of the polymerase which stops transcription
  • once mRNA has been pulled from RNA pol, it is no longer making RNA
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22
Q

What is important about translation in bacteria?

A

in bacteria, transcription and translation are very closely coupled, and translation starts before transcription is finished
- many bacterial genes are polycistronic (genes in same pathway may be transcribed as a single RNA but made into multiple proteins)

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23
Q

lac operon example of polycistronic genes

A
  • lac operon in E. coli encodes lacZ, lacY, and lacA all in one mRNA
  • unlike eukaryotic that has one mRNA per protein
  • since translation occurs before all mRNA is made, the first gene (at 5’ end) will get translated more than the last ones at the 3’ end
  • if you have good regulation, the gene product you need more of will be at 5’ end and get translated first
24
Q

how many possible codons are there?

A

64: 61 code for amino acids and 3 code for stop codons

25
Q

what are the three stop codons?

A

UAA: ochre
UGA: opal
UAG: amber

26
Q

what is AUG?

A

codon for methionine which is always the first amino acid in translation

27
Q

what makes up the 70S ribosome?

A
  • 50S: large subunit, contains a 23S and a 5S rRNA (70% RNA by mass) and 34 proteins
  • 30S: small subunit, contains a 16S rRNA (60% RNA by mass) and 21 proteins

always more RNA than protein, protein is just there to maintain stability
- S = Svedberg unit for size by density centrifugation - NOT ADDITIVE

28
Q

What is the function of 5S, 16S, and 23S rRNA?

A
  • rRNAs are the catalytic parts of ribosomes (ribozymes), proteins are there to ensure structure
  • 16S rRNA ensures accurate tRNA binding and placement of translational initiation (Shine-Dalgarno sequence)
  • 23S rRNA produces peptide bonds between amino acids and the growing protein (enzymatic)
  • 5S rRNA helps with structural integrity
29
Q

what are the APE binding sites on 23S subunit?

A

Peptidyl site binds tRNA and holds amino acid chain
Acceptor binds tRNA and holds the new amino acid to be added to the cain
Exit is a threshold and the final step before the tRNA is let go of by the ribosome

30
Q

what are tRNAs?

A
  • language translators
  • amino acids need chaperone to bring them into the growing chain unlike nucleotides which go on their own
  • tRNAs allow 3-base codon to specify an amino acid
  • anticodon loop binds the codon and the 3’ end (acceptor stem) binds the amino acid
31
Q

what are the post-transcriptional modifications on tRNAs?

A

D loop contains dihydrouracil
T loop contains ribothymidine-pseudouridine-cytosine

32
Q

what do modified anticodon bases allow for?

A

allow single tRNA to pair with multiple codons
- useful for organisms with less than 61 tRNAs
- E. coli strains have 80-100 tRNA genes but only approx. 37 discrete tRNA anticodons which can recognize all 61 codons
- mucoplasma pulmonis has 20 tRNA genes
- humans have approx. 500 tRNA genes covering approx. 50 anticodons

33
Q

What is the wobble position?

A
  • the 5’ base on anticodon (pairs with 3’ base of codon)
  • commonly modified to inosine as this can make semi-stable I:U, I:A, and I:C bonds
  • some 5’ anticodon bases can pair up with many 3’ codon bases
  • many modifications are found at the 6’ base of tRNAs (some universal, some specific to one group of eukaryotes/bacteria/archaea)
34
Q

Explain codon biases

A
  • as each organism has more of some tRNAs and less of others, having the over-represented codons in a gene will give more of exact same protein than having the “rare” codons represented
  • means that organisms may have a better affinity for a certain codon over others, or it may take less time to use a certain codon than others because there’s more of that tRNA present
  • putting gene into different organisms can have drastically different protein levels due to changes in translational efficiency to due codon bias
35
Q

How do silent mutations relate to codon bias?

A
  • may not be as silent as we think, if we switch to the more rare one it may decrease amount of protein made which would possible change the phenotype
36
Q

How does codon bias relate to translational efficiency?

A
  • 2009 paper found that in E. coli they could construct 154 different constructs of GFP that contained only synonymous/silent mutations
  • resulting GFP fluorescence was measured and varied by 250-fold
  • companies sell codon optimized gene construction as a service and claim that 1000-fold higher expression is possible in some cases
37
Q

what is the function of aminoacyl-tRNA synthetases? (aaRS)

A

Charges tRNA with the corresponding amino acid

38
Q

how do aaRS’s work?

A
  • there are generally 20 different synthetases (one for each amino acid)
  • most aaRS recognize multiple tRNAs
  • specific to all anticodons possible for a given amino acid
  • recognition is at acceptor stem and sometimes the anticodon loop
39
Q

how effective are aaRS’s?

A

only 1 in 10,000 - 100,000 tRNAs will carry the wrong amino acid because aaRS is quite selective (some have an editing domain to check tRNA has the proper amino acid before its release/can check to make sure they’re D-forms)

40
Q

How are bacterial genes regulated?

A

they are arranged in operons and steady state protein are driven by either
1. consensusness (gene promotor of variable strength and codon usage)
2. multiple genes of related function (ORFs) ranging from 2-20, where 5’ end ORFs get translated more often than 3’ ORFs

41
Q

Describe attenuation

A

if an enzyme is not needed at that time, transcription will be stopped by sensing a product of the enzyme

about 1/4 of amino acid biosynthesis genes are controlled with attenuators

42
Q

Describe generic attenuator mRNA (amino acid “X” operon) example

A

works to control at level of making mRNA
set up:
S-D sequence – AUG –2-10 codons for aa X – Region 1 – Region 2 –region 3 – poly-U
- attenuator sequence has 3 regions that are complementary to eachother, region 2 can bind region 1 or region 3, forming 2 potential stem loops (the pair depends on the rate of translation)

43
Q

How does generic attenuator work?

A

ribosome assembles at the S-D sequence and begins translating
- first amino acids incorporated are the same amino acids to be produced by the enzymes of the mRNA (senses whether it needs its own product by needing the same amino acid)

44
Q

what happens with attenuation if there’s lots of tRNA carrying aa-X?

A
  • ribosome goes quickly
  • translates region 1 while 2 and 3 gets transcribed
  • regions 2 and 3 are thus able to pair up and the stem-loop terminates transcription at poly-U site
45
Q

What happens if there aren’t many tRNA carrying aa-X?

A
  • ribosome stalls at poly-X region
  • regions 1 and 2 are able to pair up
  • region 3 stays single stranded (because region 2 is already with 1)
  • RNA pol isn’t terminated and copies and extends full mRNA
46
Q

what are riboswitches?

A

RNA sensors that can fold to produce a structure capable of undergoing conformational change

47
Q

What are 2 examples of riboswitches?

A

-RNA thermometers that change structure based on temperature
- small-molecule binding aptamers that change structure based on presence/absence of the target compound

48
Q

what effects can riboswitches have?

A

Can be transcriptional or translational
- prevent or allow binding of Rho to terminate transcription prematurely
- prevent or allow 16S rRNA to bind S-D site
- Produce a ribozyme that cleaves/destroys itself if not needed

49
Q

Example of RNA thermometer in pathogenicity

A
  • A “fourU” RNA-therm from salmonella is located in the 5’ UTR of the mRNA for a heat shock protein called AgsA
  • S-D sequence pairs with fourU site and forms a stable stem loop at lower temperatures which prevents translation because small ribosome can’t bind S-D
  • at higher temperatures it melts and makes S-D accessible which suppresses protein aggregation (like when infecting a mammalian host)
  • another fourU sequence controls virulence expression in Vibrio cholerae
50
Q

What is the function of the Lac operon?

A
  • transcriptional repression and activation of lac operon results in control at level of DNA binding
  • lactose is disaccharide of glucose and galactose
  • Lac operon determines when β-galactosidase breaks lactose down
  • using lactose is less efficient than glucose because it requires additional enzymes to break it down
51
Q

when is expression of enzymes to import and break down lactose undesirable?

A
  • when glucose is already present
  • when there isn’t any lactose available
  • only if both conditions are false does the lac operon get fully expressed
  • there is 50,000-fold expression increase from repressed to activated
52
Q

What is the overall setup of lac operon?

A

lacl – CAP – P – O – lacZ – lacY – lacA

  • polycistronic message lacZ (β-galactosidase), lacY, lacA is controlled partialy by lacI gene
  • LacI encodes a repressor that bind and shuts off the lac operon (lacA/Y/A)
  • CAP is where “on” binding occurs
  • P is promoter region
  • O is operator: DNA binding sequence specific to the repressor (where “off” binding occurs)
53
Q

What does the LacI gene do?

A
  • produces Lac repressor
  • is a weak σ₇₀ promoter leading to low levels of mRNA and protein in cell (not every base is part of the consensus sequence)
  • some promoter mutants (such as LacI^Q) overexpress repressor so lac operon is always off
54
Q

Lac repressor from Lac I

A

-tetramer of 2 (out of 3 possible) closely located regions of DNA
- binding blocks access to the promoter by forcing the DNA to loop out (dimers pull two ends of the DNA together to form a loop)
- RNA polymerase can’t displace the repressor so can never access the promoter to initiate transcription
- if all 3 operators are deleted, we can never turn the gene off (don’t necessarily turn it on either)
- if all 3 operons are present, there is much better reduction in expression

55
Q

What happens when Lac Repressor binds Allolactose?

A
  • β-galactosidase expressed at minimal levels converts lactose to allolactose
  • allolactose binds each lac repressor monomer in the tetramer which destabilizes the tetramer which unlocks the lac repressor allowing the closed-loop of promoter DNA to be bound by RNA polymerase
56
Q

How does Lac activation work to turn things on?

A
  • to get full expression, E. coli needs more than lactose (absence of glucose is also necessary)
  • adenylase cyclase converts ATP to cyclic AMP
  • does not work well and has low activity when glucose is abundant (acts as sensor)
  • works better and has higher activity when glucose is low
  • high glucose = low cAMP
  • low glucose = high cAMP
  • cAMP binds to catabolite activator protein (CAP) which is transcriptional activator
    -CAP binding sites are 5’ to the lac promotor (and 200 other genes)
    -TGTTA (run of other bases) TCACA is the basic consensus sequence
57
Q

how is RNA pol recruited by cAMP/CAP?

A
  • if no cAMP because lots of glucose present, CAP blocks DNA 2 binding sites
  • if cAMP accumulates and binds CAP, conformation of CAP exposes DNA binding sites so CAP sticks to major groove of DNA
  • CAP also has activation domain which recruits RNA pol to make sure RNA pol starts transcription more often
  • CAP kind of acts like a σ factor

BMSC320 (9 decks)

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