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PHGY - Midterm > MICR - Week 3 > Flashcards

MICR - Week 3 Flashcards

1
Q

What species of bacteria is used to make recombinant insulin?

A

E.coli

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2
Q

Describe pathogenesis in terms of translation/transcription

A

Certain proteins are needed to colonize the host, but if not in host don’t need them so don’t prescribe them

Will sense things like:
- temp
- nutrients
- immune system

Then make things like:
- to attach to cell (pili)
- to evade the immune sys
- to kill host cell

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3
Q

A promotor region is part of

A

transcription

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4
Q

A promoter and terminator sequence are used in transcription, where are they located?

A

Promoter = 5’ end
Terminator = 3’ end

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5
Q

Define: operon

A

Operon: multiple genes that share a promoter

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6
Q

What does the transcription of the operon make?

A

Transcription of the operon makes polycistronic mRNA

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7
Q

Transcription has 3 stages - describe

A

Initiation:
1) Sigma factor helps RNA pol recognize promoter
2) RNA pol binds DNA between -35 and -10 region forming a closed complex
3) RNA pol unwinds DNA forming an open complex
4) RNA synthesis begins and sigma factor falls off

Elongation:
1) NTPs are added to 3’ end of RNA-DNA hybrid
2) DNA is rewound as RNA pol progresses

Termination:
There are 2 ways:

= Factor dependent termination
- Rho factor separate RNA-DNA hybrd

= Intrinsic terminators
- inverted repeats for a hairpin/stem loop, with a poly U tail
- the U A bond is weak, so RNA/DNA dissociates and R pol falls off

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8
Q

Define: constitutive genes

A

Constitutive genes = genes that are always transcribed

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9
Q

Name 2 ways transcription can be regulated (general)

A

1) DNA binding proteins (initiation)
2) mRNA structure (elongation, termination)

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10
Q

How does bacteria regulate initiation

A

Sigma Factors - Regulate Initiation
- bacteria uses multiple promoters, and RnaP requires a sigma factor to bind
- diff sigma factors bind diff promoters
- Thus sigma factors control which genes are transcribed, and a change in the level of sigma factors present change the level of transcription

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11
Q

Regulating Initiation - Repressors
Describe

A
  • repressor proteins block initiation
  • when a repressor binds, rna p can’t transcribe the gene
  • repressors bind the operator region, if a ligand binds them it causes a conformational changing its shape so it can’t bind
  • is under negative transcriptional control
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12
Q

Negative Control of Inducible Genes
- Describe

A
  • some repressor controlled genes are inducible
    Inducible: repressor binding to operator turns off transcription
  • Induce ligand stops repressor from binding to operator, turns transcription on
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13
Q

When a ligand binds a repressor, does it turn on or off transcription?

A

It turns on transcription

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14
Q

When a co-repressor binds a repressor, does it turn on or off transcription?

A

Co-repressors = a ligand that helps repressors bind DNA
Thus, turns off transcription

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15
Q

Describe the regulation of the Trp operon

A
  • TrpR = repressor
  • Trp = co-repressor

High Trp = don’t need more /trp, so Trp binds TrpR to turn off transcription

Low Trp = since Trp is low, it can’t act as a co-represspr thus the operon is transcribed

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16
Q

What example was used for bifunctional activator/repressor?

A

AraC

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17
Q

Attenuation - Define

A

Attenuation: ribosomal stalling, impacts transcription

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18
Q

What causes attenuation?

A

Low aa abundance causes the ribosome to stall, stalling increases transcription of the operon

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19
Q

Tell me about the leader region?

A

Leader region: ex. TrpL

Location: between the operator and the structural genes
Purpose: encodes for a leader peptide which has no purpose, is just there for attenuation

If TrpL is not being translated:
- 1 and 2, 3 and 4 base pair
- poly U and hairpin form loop for terminator
- RNA P stops transcribing DNA at the poly U tract

If Trp is low, ribosome stalls at Trp codons:
- blocks 1 and 2 from pairing
- 2 and 3 form an antiterminator
- terminator is not formed
- RNAP transcribes genes after DNA poly T tract

If Trp is high, ribosome translates Trp codons, but stalls at TrpL codon:
- blocks pairing of 2 and 3
- 3 and 4 pair forming terminator
- RNAP does not transcribe genes after poly T tract

20
Q

Define Riboswitch

A

Riboswitch: mRNA leader region that can adopt 2 different conformations affecting transcription

21
Q

What are the 2 conformations of riboswitches

A

Switch is normally off
- terminator stops at poly T
- can be turned on by metabolite/ligand
- binding promotes formation of anti-terminator, which prevents terminator from forming, thus transcription is not stopped at poly T

Switch is normally on
- transcription does not stop at poly T
- turned off by metabolite
- binding promotes formation of anti-antiterminator
- terminator forms, transcription stops at ply T

22
Q

What was one of the first antibiotics discovered?

A

Streptomycin

23
Q

Streptomycin
What is it effective against:

A

Streptomycin
What is it effective against: Gram negatives

24
Q

Define: Aminoglycoside

A

Aminoglycoside: antibiotic that targets the ribosome

25
Q

Where does the ribosome assemeble?

A

The ribosome assembles at the ribosome binding site (RBS) - the Shine-Dalgarno sequence

26
Q

Where does the ribosome start and stop?

A

Start = start codon
Stop = stop codon

27
Q

What does tRNA have at 3’ end?

A

Has aa attached to 3’ acceptor end
- aminoacyl-tRNA synthetase

28
Q

Aminoacyl-tRNA Synthetase
What are its features

A

Anticodon binding domain
- recognizes tRNA anticodon
- makes sure its the right reaction

Catalytic domain:
- attaches aa to tRNA

  • AA must be activated by ATP
29
Q

What type of bind links aa to tRNA

A

ester bond

30
Q

What is the bacterial ribosome made of?

A

rRNA and proteins

31
Q

Ribosome - function

A

Ribosome: forms peptide bonds between aa’s on tRNA
- ribosome helps tRNA anticodon bind to mRNA codons

32
Q

tRNA function

A

tRNAs deliver aa to ribosome

33
Q

Describe all the steps of transcription

A

Initiation
1) Nucleotide in 16S rRNA (30S subunit) binds to mRNA RBS
2) IF3 binds to 30s subunit and ensures 50S subunit does not bind too soon
3) Once mRNA is recruited to 30s, IF2 helps deliver fMet
4) IF1 binds displacing IF3
5) Forms 30S initiation complex
6) 50S subunit joins 30s initiation complex (Needs GTP)
7) IFs are released

Elongation
1) Elongation factor EF-TU delivers tRNA to A site (GTP)
2) tRNA anticodon binds to codon
3) Peptide bind formed between amino acids in A site, and P site
NOTE: 1st aa comes into P site, after that they come into A site

Elongation - Translocation
1) EF-TU and GTP help the ribosome navigate along the mRNA sequence
2) mRNA advances thru the ribosome
3) Uncharged tRNA is in the E site exits
4) t-RNA with peptide moves to P site
5) Another tRNA is delivered to A site, elongation continues

Termination
1) Ribosome stall at stop codon
- UAA, UGA, UAG
- no tRNA
2) Stop codon is recognized by release factors
3) Release factors hydrolyze polypeptide from tRNA
4) Translation complex dissociates

34
Q

Why is the ribosome a good target for antibiotics

A

Bc the bacterial ribosome is different from the eukaryotic one

35
Q

How do Tetracyclines function

A

Tetracyclines
- bind to rRNA in 30s subunit (A site)
- occupy the binding site of tRNA anticodons
- block delivery of tRNA

36
Q

Define - Bacteriostatic

A

Bacteriostatic: inhibits growth without killing
- growth resumes if removed

37
Q

How do Macrolides function?

A
  • Growing poly-p extend thru exit channel
  • Macrolides bind to 50S subunit blocking exit tunnel
  • prevents elongation of nascent poly-p chain (NC)
38
Q

How do Lincosamides function?

A
  • They bind to the A site of t the 50S subunit
  • This disrupts the positioning of A site tRNA in peptidyl transferase center (PTC)
  • blocking peptide bond formation
39
Q

Give and example of a Lincosamide

A

Clindamycin

40
Q

What are some antibiotic resistance mechanisms?

A
  • can limit the amount of antibiotic in cytoplasm
  • can prevent the antibiotic from binding to ribosome by:
    - modifying ribosome
    - modifying antibiotic
41
Q

What do ERM groups do?

A

They change ribosome structure but not function as a means of antibiotic resistance

42
Q

Translational Riboswitches
define
What are the 2 types - Describe

A

Riboswitch: secondary structure in mRNA leader region (before genes)

1) Sequester - off by default
- RBS is unavailable
- turned ON by ligands
- ligand binding forms anti-sequester

2) Anti-Sequester - on by default
- RBS is available
- turned off by ligands
- ligand binding forms anti-antisequester making RBS unavailable

43
Q

What do sRNAs regulate?

A

Small RNAs regulate translation

44
Q

What are sRNAs ( 2 types) , What are they capable of?

A

sRNAs are non-coding RNAs

Cis-encoded = sRNA is produced from the same DNA as mRNA

Trans-encoded = sRNA is produced from some other DNA

Binding to …
- mRNA impacts translation
- RBS blocks initiation
- gene blocks elongation
- leader region prevents a sequester from forming = RBS more accessible = translation increases
- mRNA can target degradation by ribonuclease (translation decreases)

45
Q
A

PHGY - Midterm (10 decks)

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