MICR - Week 3 Flashcards
What species of bacteria is used to make recombinant insulin?
E.coli
Describe pathogenesis in terms of translation/transcription
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
A promotor region is part of
transcription
A promoter and terminator sequence are used in transcription, where are they located?
Promoter = 5’ end
Terminator = 3’ end
Define: operon
Operon: multiple genes that share a promoter
What does the transcription of the operon make?
Transcription of the operon makes polycistronic mRNA
Transcription has 3 stages - describe
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
Define: constitutive genes
Constitutive genes = genes that are always transcribed
Name 2 ways transcription can be regulated (general)
1) DNA binding proteins (initiation)
2) mRNA structure (elongation, termination)
How does bacteria regulate initiation
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
Regulating Initiation - Repressors
Describe
- 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
Negative Control of Inducible Genes
- Describe
- 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
When a ligand binds a repressor, does it turn on or off transcription?
It turns on transcription
When a co-repressor binds a repressor, does it turn on or off transcription?
Co-repressors = a ligand that helps repressors bind DNA
Thus, turns off transcription
Describe the regulation of the Trp operon
- 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
What example was used for bifunctional activator/repressor?
AraC
Attenuation - Define
Attenuation: ribosomal stalling, impacts transcription
What causes attenuation?
Low aa abundance causes the ribosome to stall, stalling increases transcription of the operon
Tell me about the leader region?
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
Define Riboswitch
Riboswitch: mRNA leader region that can adopt 2 different conformations affecting transcription
What are the 2 conformations of riboswitches
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
What was one of the first antibiotics discovered?
Streptomycin
Streptomycin
What is it effective against:
Streptomycin
What is it effective against: Gram negatives
Define: Aminoglycoside
Aminoglycoside: antibiotic that targets the ribosome
Where does the ribosome assemeble?
The ribosome assembles at the ribosome binding site (RBS) - the Shine-Dalgarno sequence
Where does the ribosome start and stop?
Start = start codon
Stop = stop codon
What does tRNA have at 3’ end?
Has aa attached to 3’ acceptor end
- aminoacyl-tRNA synthetase
Aminoacyl-tRNA Synthetase
What are its features
Anticodon binding domain
- recognizes tRNA anticodon
- makes sure its the right reaction
Catalytic domain:
- attaches aa to tRNA
- AA must be activated by ATP
What type of bind links aa to tRNA
ester bond
What is the bacterial ribosome made of?
rRNA and proteins
Ribosome - function
Ribosome: forms peptide bonds between aa’s on tRNA
- ribosome helps tRNA anticodon bind to mRNA codons
tRNA function
tRNAs deliver aa to ribosome
Describe all the steps of transcription
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
Why is the ribosome a good target for antibiotics
Bc the bacterial ribosome is different from the eukaryotic one
How do Tetracyclines function
Tetracyclines
- bind to rRNA in 30s subunit (A site)
- occupy the binding site of tRNA anticodons
- block delivery of tRNA
Define - Bacteriostatic
Bacteriostatic: inhibits growth without killing
- growth resumes if removed
How do Macrolides function?
- Growing poly-p extend thru exit channel
- Macrolides bind to 50S subunit blocking exit tunnel
- prevents elongation of nascent poly-p chain (NC)
How do Lincosamides function?
- 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
Give and example of a Lincosamide
Clindamycin
What are some antibiotic resistance mechanisms?
- can limit the amount of antibiotic in cytoplasm
- can prevent the antibiotic from binding to ribosome by:
- modifying ribosome
- modifying antibiotic
What do ERM groups do?
They change ribosome structure but not function as a means of antibiotic resistance
Translational Riboswitches
define
What are the 2 types - Describe
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
What do sRNAs regulate?
Small RNAs regulate translation
What are sRNAs ( 2 types) , What are they capable of?
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)
