Lesson 2.2 - DNA & Inheritance Flashcards
Flow of Information / Gene Expression in Bacteria & Archaea
DNA (genotype) = mRNA = Protein (phenotype)
(3) Phases of Transcription Overview
- Initiation
- RNA poly. recognize DNA sites; binds template strand
- Elongation
- RNA poly unwinds DNA; adds nucleotides
- Termination
- Ends; Free mRNA
What is RNA polymerase?
- Identifies promoter
- Multiple subunit enzyme; makes mRNA from DNA
- de novo synthesis: no primer needed
What does RNA polymerase bind to?
-10 and -35 sites
What is a promoter?
- Binding site for RNA polymerase; immediately preceds gene
- Determines which DNA strand is template
What are (3) key sequences of a promoter?
- Start point; first based transcribed (+1)
- A-T rich sequence; TATA box (-10)
- T-G rich sequence (-35)
Define upstream and downstream sequences/
- Upstream sequences: before start point (-)
- Downstream sequences: after start point (+)
What is the 5’ UTR?
Important regulatory function; starts at +1 and ends 1 nucleotide before start codon
What is the distance between -10 and -35 sequence? Why?
16 - 18 bp. Size matters. Connects RNA poly. to DNA
What are the (2) parts of the holoenzyme (RNA polymerase)?
- 6 polypeptides
- Core enzyme (αIIββ’ω)
- Binding and mRNA synthesis
- Sigma (σ) factor
- Promoter recognition
- Core enzyme (αIIββ’ω)
What do the α subunits do?
Help assemble core and contact promoter
What do the β and β’ subunits do?
Form main channel through which DNA passes, 2o channel for rNTPs and the exit channel for mRNA
What do the ω subunits do?
Involved in enzyme assembly and regulation
Function of sigma (σ) factors
- Control binding to DNA by recognizing -10 & -35
- Released after transcription starts
- Ensures bacterial RNA poly. binds at promoters & initiates transcription
- σ70 = housing keeping
- Sporulation; special sigma factors
- σE, σF, σG, σK
What does the core enzyme do?
Can synthesize DNA on a template in vitro, but can’t recognize promoters; has general affinity to DNA
What occurs during elongation (transcription)?
- RNA poly reads template 3’ to 5’
- Runs antiparallel
- mRNA synthesized in 5’ to 3’ (OH group)
- Adding rNTPs (ATP, CTP, GTP, UTP)
- Form phosphodiester bonds
How large is the ‘transcription bubble?’
Where RNA synthesis takes place; encompasses 12-14 base pairs
Positive supercoiling
Right-handed, double-helical conformation of DNA is twisted even tighet (right-handed twisting); helix begins to knot
Negative supercoiling
Twisting against helical conformation (left-handed twisting); straightens, relaxes DNA
RNA poly. generates ____ supercoils ahead and _____ supercoils behind
(+1); (-1)
Gyrase
Creates negative supercoils via ds breaks
Topoisomerase I
Removes (-) supercoils via ss breaks; creates positive supercoil
What occurs during termination (transcription)?
- Termination sequence is reached
- Hairpins form due to G-C
- Slows down, dislodges RNA polu
- Intrinsic terminator
What are rho-dependent terminators?
- rho (ρ) factor binds at rut (rho utalization) at 5’ end of new mRNA
- C-rich and G-poor region; no hairpins
- Rho moves along mRNA, catches up to RNA poly, unwinds DNA/RNA; RNA poly falls off
-
rut sites found near intrinsic termination site
- Not restricted to 5’ of mRNA
Polycistronic
Bacteria and Archaea; multiple genes on mRNA
Monocistronic
Eukaryotes; have 5’ cap and poly-A tail
mRNA organized in groups of (3) …
3 ribonucleotides = codons
Start codons
- 83% AUG
- 14% GUG
- 3% UUG
Stop codons
UAG, UAA, UGA
Three sites in translation
E, P, A
What occurs during initiation (translation) in Bacteria?
- 30S subunit binds to RBS at 5’ end of mRNA
- AG rich sequence complementary to 3’ end of 16S rRNA
- tRNAf-Met enters partial P site before large subunit binds; formyl is removed
- Large ribosomal subunit joins (initiation factos help)
- Hydrolysis of GTP to GDP
In Bacteria, special initiator tRNAs recognize…
- …start codons
- Bacteria use N-formylmethionine
- Archaea & Eukaryotes use regular methionine
- Bacteria use regular methionine during elongation
What is the Shine-Dalgarno (SD) sequence?
Ribosomal binding site in bacterial and archaeal mRNA, located around 8 bases upstream of start codon (AUG)
What occurs during elongation (translation)?
- Elongation factors help H-binding b/t codon under site A and anticodon of next tRNA
- 2 GTP required
- Peptidyl transferase ribozyme forms peptide bond b/t Met & Pro
- Uncharged tRNA in P site moves to E site; A site is available for next charge tRNA
What occurs during termination (translation)?
- Release factor bind to stop codon, polypeptide freed
- Elongation complex dissociated
Polysome
Multiple ribosomes on single mRNA; allows organism to make lots of proteins
Open reading frame
Region of mRNA that codes for product
Flouroquinolones
Bind to DNA gyrase and topoisomerase IV
e.g. Ciprofloxacin
Topoisomerase IV
Relaxes positive DNA supercoils (overtwisting) during DNA replication and transcription; inhibition is lethal
DNA gyrase
Relaxes positive supercoils (over-twisting) ahead of DNA polymerase; inhibition is lethal
Antibiotic that inhibit transcription
Rifamycins
Rifamycin
Binds to ß subunit (catalytic) of bacterial DNA; prevents/interferes with transcription
e.g. rifabutin
Antibiotics that disrupt translation (3 classes)
- Chloramphenicol, macrolides, and lincosamides
- 50S ribosomal subunit; peptide bond formation
- Aminoglycosides
- 30S ribosomal subunit; proofreading
- Tetracyclines
- 30S ribosomal subunit; tRNA binding
Aminoglycosides
- 30S ribosomal subunit
- Ribosome misreads codons
- e.g. gentamycin, neomycin, streptomycin
Tetracyclines
- 30S ribosomal subunit (reversibly)
- Prevents attachment of incoming aminoacyl-tRNA
- site A affected
- e.g. doxycycline, minocycline, tetracycline
Oxazolidinones
- 50S ribosomal subunit (P site)
- Inhibits translocation
- e.g. linezolid, tedizolid
Chloramphenicol
- 50S ribosomal subunit (reversibly)
- Disrupt peptidyl transferase activity
- Transfer of AAs to peptide chain; bond formation
Macrolides
- 50S ribosomal subunit
- Binds & partially blocks peptide exit tunnel, site E
- Semi-selective; some peptides can navigate blockage
- Modulator of translation
- e.g. azithromycin, clarithromycin, erythromycin
