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
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
Rifamycin
Binds to ß subunit (catalytic) of bacterial DNA; prevents/interferes with transcription
e.g. rifabutin
