Lesson 2.2 - DNA & Inheritance

Question 1

Flow of Information / Gene Expression in Bacteria & Archaea

Answer 1

DNA (genotype) = mRNA = Protein (phenotype)

Question 2

(3) Phases of Transcription Overview

Answer 2

• Initiation
  
  • RNA poly. recognize DNA sites; binds template strand
• Elongation
  
  • RNA poly unwinds DNA; adds nucleotides
• Termination
  
  • Ends; Free mRNA

Question 3

What is RNA polymerase?

Answer 3

• Identifies promoter​
• Multiple subunit enzyme; makes mRNA from DNA
• de novo synthesis: no primer needed

Question 4

What does RNA polymerase bind to?

Answer 4

-10 and -35 sites

Question 5

What is a promoter?

Answer 5

• Binding site for RNA polymerase; immediately preceds gene
• Determines which DNA strand is template

Question 6

What are (3) key sequences of a promoter?

Answer 6

• Start point; first based transcribed (+1)
• A-T rich sequence; TATA box (-10)
• T-G rich sequence (-35)

Question 7

Define upstream and downstream sequences/

Answer 7

• Upstream sequences: before start point (-)
• Downstream sequences: after start point (+)

Question 8

What is the 5’ UTR?

Answer 8

Important regulatory function; starts at +1 and ends 1 nucleotide before start codon

Question 9

What is the distance between -10 and -35 sequence? Why?

Answer 9

16 - 18 bp. Size matters. Connects RNA poly. to DNA

Question 10

What are the (2) parts of the holoenzyme (RNA polymerase)?

Answer 10

• 6 polypeptides
  
  • Core enzyme (α_IIββ’ω)
    
    • Binding and mRNA synthesis
  • Sigma (σ) factor
    
    • Promoter recognition

Question 11

What do the α subunits do?

Answer 11

Help assemble core and contact promoter

Question 12

What do the β and β’ subunits do?

Answer 12

Form main channel through which DNA passes, 2^o channel for rNTPs and the exit channel for mRNA

Question 13

What do the ω subunits do?

Answer 13

Involved in enzyme assembly and regulation

Question 14

Function of sigma (σ) factors

Answer 14

• 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
    
    • σ^E, σ^F, σ^G, σ^K

Question 15

What does the core enzyme do?

Answer 15

Can synthesize DNA on a template in vitro, but can’t recognize promoters; has general affinity to DNA

Question 16

What occurs during elongation (transcription)?

Answer 16

• 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

Question 17

How large is the ‘transcription bubble?’

Answer 17

Where RNA synthesis takes place; encompasses 12-14 base pairs

Question 18

Positive supercoiling

Answer 18

Right-handed, double-helical conformation of DNA is twisted even tighet (right-handed twisting); helix begins to knot

Question 19

Negative supercoiling

Answer 19

Twisting against helical conformation (left-handed twisting); straightens, relaxes DNA

Question 20

RNA poly. generates ____ supercoils ahead and _____ supercoils behind

Answer 20

(+1); (-1)

Question 21

Gyrase

Answer 21

Creates negative supercoils via ds breaks

Question 22

Topoisomerase I

Answer 22

Removes (-) supercoils via ss breaks; creates positive supercoil

Question 23

What occurs during termination (transcription)?

Answer 23

• Termination sequence is reached
• Hairpins form due to G-C
  
  • Slows down, dislodges RNA polu
  • Intrinsic terminator

Question 24

What are rho-dependent terminators?

Answer 24

• 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

Question 25

Polycistronic

Answer 25

Bacteria and Archaea; multiple genes on mRNA

Question 26

Monocistronic

Answer 26

Eukaryotes; have 5' cap and poly-A tail

Question 27

mRNA organized in groups of (3) ...

Answer 27

3 ribonucleotides = codons

Question 28

Start codons

Answer 28

* 83% AUG * 14% GUG * 3% UUG

Question 29

Stop codons

Answer 29

UAG, UAA, UGA

Question 30

Three sites in translation

Answer 30

E, P, A

Question 31

What occurs during initiation (translation) in Bacteria?

Answer 31

* 30S subunit binds to RBS at 5' end of mRNA * AG rich sequence complementary to 3' end of 16S rRNA * tRNA^f-Met enters partial P site before large subunit binds; formyl is removed * Large ribosomal subunit joins (initiation factos help) * Hydrolysis of GTP to GDP

Question 32

In Bacteria, special initiator tRNAs recognize...

Answer 32

* ...start codons * Bacteria use N-formylmethionine * Archaea & Eukaryotes use regular methionine * Bacteria use regular methionine during elongation

Question 33

What is the Shine-Dalgarno (SD) sequence?

Answer 33

**Ribosomal binding site** in bacterial and archaeal mRNA, located around 8 bases upstream of start codon (AUG)

Question 34

What occurs during elongation (translation)?

Answer 34

* 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

Question 35

What occurs during termination (translation)?

Answer 35

* Release factor bind to stop codon, polypeptide freed * Elongation complex dissociated

Question 36

Polysome

Answer 36

Multiple ribosomes on single mRNA; allows organism to make lots of proteins

Question 37

Open reading frame

Answer 37

Region of mRNA that codes for product

Question 38

Flouroquinolones

Answer 38

Bind to DNA gyrase and topoisomerase IV e.g. Ciprofloxacin

Question 39

Topoisomerase IV

Answer 39

Relaxes positive DNA supercoils (overtwisting) during DNA replication and transcription; inhibition is lethal

Question 40

DNA gyrase

Answer 40

Relaxes positive supercoils (over-twisting) ahead of DNA polymerase; inhibition is lethal

Question 41

Antibiotic that inhibit transcription

Answer 41

Rifamycins

Question 42

Rifamycin

Answer 42

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

Question 43

Antibiotics that disrupt translation (3 classes)

Answer 43

* Chloramphenicol, macrolides, and lincosamides * 50S ribosomal subunit; peptide bond formation * Aminoglycosides * 30S ribosomal subunit; proofreading * Tetracyclines * 30S ribosomal subunit; tRNA binding

Question 44

Aminoglycosides

Answer 44

* 30S ribosomal subunit * Ribosome misreads codons * e.g. gentamycin, neomycin, streptomycin

Question 45

Tetracyclines

Answer 45

* 30S ribosomal subunit (reversibly) * Prevents attachment of incoming aminoacyl-tRNA * site A affected * e.g. doxycycline, minocycline, tetracycline

Question 46

Oxazolidinones

Answer 46

* 50S ribosomal subunit (P site) * Inhibits translocation * e.g. linezolid, tedizolid

Question 47

Chloramphenicol

Answer 47

* 50S ribosomal subunit (reversibly) * Disrupt peptidyl transferase activity * Transfer of AAs to peptide chain; bond formation

Question 48

Macrolides

Answer 48

* 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