MICROBIOLOGY TEST 3

Like bacteria, archaea have ______________.

• Polygenic operons,

• A single circular chromosome

• Cells lacking a nuclear membrane

Many pharmaceutical drugs specifically inhibit transcription in bacteria but not archaea or eukarya. Why would drugs that inhibit transcription only affect Bacteria and not Archaea even though they are both prokaryotes?

Archaea and Eukarya have very similar RNA polymerases that are different frombacterial RNA polymerases.

All sigma factors compete for the same number of core RNA polymerase enzymes.

TRUE

During transcription, if the coding strand of DNA has the sequence 3' TCTAGGACT 5', what will the sequence of the transcribed RNA be?

5' UCAGGAUCU 3'

The termination of transcription can be caused by which of the following? Select all that apply.

• Rho factor

• Transcription of inverted repeats

What is the sequence of amino acids encoded by strand of DNA template 3' CGTACGTTCTTA 5'

alanine -cysteine -lysine -asparagine

You are analyzing a series of mutants that have defects in either transcription or translation. Which of the following mutations would you predict to impact translation directly without affecting transcription?

• 30S subunit

• 16S rRNA

• Shine-Dalgarno sequence

What is the main difference between a mRNA and tRNA?

Only mRNA gets be translated to amino acid sequence

Which of the following is NOT a fate for a newly made bacterial proteins (nascent proteins).

Translated back to mRNA so that they can be recycled

In a PCR reaction, you mixed all the necessary ingredients. You used DNA polymerase from a bacterium Thermus aquaticus, and the DNA template from E. coli. The sequence of the newly made DNA would look like _____.

E. coli DNA

genetic code

set of rules that determines how a nucleotide sequence is converted to an amino acid sequence of a protein

Central Dogma

1. DNA → RNA → Protein

   Steps

   1. Replication = DNA copies itself

   2. Transcription = DNA → RNA

   3. Translation = RNA → Protein

Central Dogma Exception

• Retroviruses (HIV)

  • RNA → DNA using reverse transcriptase

• Some viruses have RNA genomes

Genes

Basic unit of heredity; codes for protein (monocistronic) or part of operon (polycistronic).

Genome

All genetic material. Prokaryotes: usually 1 circular dsDNA chromosome + plasmids. Eukaryotes: larger, linear, more noncoding DNA (>90%).

Operon

Genes coordinately regulated (e.g., thrABC).

Monocistronic

One gene → one protein

Polycistronic

Several genes → several proteins

(Bacteria commonly use this.)

Nucleoid

Bacterial DNA storage region

DNA packaged by:

• HU proteins

• H-NS proteins

Griffith discovered?

Transformation

A

T

G

C

DNA helicase?

DnaB

Main DNA polymerase?

DNA Pol III

Removes RNA primers?

DNA Pol I

Joins fragments?

Ligase

Replication starts at?

oriC

Replication ends at?

ter

Lagging strand fragments?

Okazaki fragments

PCR polymerase?

Taq polymerase

Two transcription termination methods?

Rho-dependent and Rho-independent

Why are transcription and translation coupled in bacteria?

A: No nucleus

Griffith Experiment (1928)

• Live R + Heat-killed S → Mouse dies

• R cells absorbed DNA from dead S cells.

• Transformation

  • Uptake of external DNA causing genotype and phenotype change.

Components of Nucleotide

1. Phosphate

2. Deoxyribose sugar

3. Nitrogenous base

Bases

Purines:

• A

• G

Pyrimidines:

• T

• C

Chargaff's Rules

Base pairing

• A-T = 2 hydrogen bonds

• G-C = 3 hydrogen bonds

Watson & Crick Model

Features

• Double helix

• Antiparallel strands

• Sugar-phosphate backbone outside

• Bases inside

Important

More G-C = stronger DNA

Because: 3 H-bonds > 2 H-bonds

DNA Denaturation

• High temperatures: 50-90°C

• Cause: Hydrogen bonds break

• Result: Double-stranded DNA becomes single-stranded

Genome Organization Prokaryotes

• Small genomes

• Mostly coding DNA

• Circular chromosome

• Plasmids present

Genome Organization Eukaryotes

• Large genomes

• Linear chromosomes

• Histones

• Lots of noncoding DNA (>90%)

Plasmids

Characteristics

• Circular DNA

• Extrachromosomal

• Replicate independently

Carry genes for

• Antibiotic resistance

• Pathogenicity

• Symbiosis

Acquired by

• Horizontal gene transfer

Negative Supercoils

Underwound

Most common

Positive Supercoils

Overwound

Common in thermophilic archaea

Why?

More stable at high temperatures

Type I Topoisomerase

Cuts:
One strand

Function:
Relieves supercoils

Type II Topoisomerase

Cuts:
Both strands

Function:
Introduces negative supercoils

DNA Gyrase

Type II enzyme

Function:
Creates negative supercoils

Target of:
Quinolones

Why good antibiotic target?

Humans lack DNA gyrase

DNA Replication

Semiconservative

Each daughter DNA:

1 old strand
1 new strand

Bidirectional

Starts at origin and moves both directions

Replication Enzymes

DnaA, DnaB, SSBP, Primase, DNA Pol III, DNA Pol, DNA Ligase, Topoisomerase IV, Tus, XerCD

DnaA

Initiates replication at oriC

DnaB

Helicase
Unwinds DNA

SSBP

Keeps strands apart

Primase

Makes RNA primer

DNA Pol III

Main replication enzyme

Adds nucleotides

DNA Pol I

Removes RNA primers
Replaces with DNA

DNA Ligase

Joins DNA fragments

Topoisomerase IV

Separates daughter chromosomes

Tus

Stops replication at ter sites

XerCD

Resolves catenanes

Leading Strand

5' → 3'

Lagging Strand

Discontinuous synthesis

Produces:

Okazaki Fragments

Joined by ligase

. PCR

Polymerase Chain Reaction

Purpose:
Amplify DNA

PCR Steps

1. Denaturation

95°C

DNA separates

2. Annealing

55°C

Primers bind

3. Extension

72°C

Taq polymerase copies DNA

Enzyme

Taq Polymerase

Source:
Thermus aquaticus

Heat resistant

Transcription

DNA → RNA

Performed by:
RNA Polymerase

INITIATION- ELONGATION-TERMINATION-RHO-independent

Initiation

RNA polymerase binds promoter

Sigma factor helps

Elongation

RNA synthesized

~45 bases/sec

Termination

Rho-dependent

Uses Rho protein

Rho-independent

Stem-loop forms

RNA → Protein- Occurs on ribosomes

Initiation

Elongation

Termination

Stop codon reached

Coupled Transcription & Translation

Bacteria

Possible

Why?

No nucleus

Eukaryotes

Impossible

Why?

Transcription occurs in nucleus

Translation occurs in cytoplasm

Silent

No amino acid change

Missense

Different amino acid

Nonsense

Creates stop codon

Frameshift

Insertion/deletion
Shifts reading frame

Most severe

Horizontal Gene Transfer

Transformation

Free DNA uptake

Transduction

Phage transfer

Conjugation

Plasmid transfer

How Resistance Develops

Vertical Evolution

Mutations

Passed to offspring

1. Modify Target

Ribosome changes

Drug can't bind

2. Destroy Drug

β-lactamase destroys penicillins

3. Modify Drug

Chemical groups added

Drug inactivated

4. Efflux Pumps

Pump drug out

Cause multidrug resistance

Azoles

Block ergosterol synthesis

Amphotericin B

Creates membrane pores

Caspofungin

Blocks fungal cell wall synthesis

Griseofulvin

Disrupts mitosis

Broad Spectrum

Targets many species

Gram + and Gram -

Example:
Tetracycline

Narrow Spectrum

Targets specific organisms

Example:
Isoniazid

Bactericidal

Kills bacteria

Bacteriostatic

Stops growth

Relies on immune system

Effective Antibiotic Requirements

Must:

1. Harm microbe

AND

2. Not harm host

MIC

Minimum Inhibitory Concentration

Lowest concentration preventing growth

MBC (MLC)

Minimum Bactericidal Concentration

Lowest concentration killing bacteria

Kirby-Bauer Test

Procedure

1. Bacterial lawn

2. Antibiotic disks added

3. Incubate

4. Measure zones

Zone of inhibition

Clear area around disk

Larger zone = generally more susceptibility

Cell Wall

Penicillin (β-lactam)

Blocks:
Transpeptidase

Prevents cross-linking

Vancomycin

Binds:
D-Ala-D-Ala

Prevents cell wall synthesis

Bacitracin

Blocks:
Bactoprenol

Cell Membrane

Gramicidin

Forms ion channels

Polymyxin (Colistin)

Acts like detergent

Destroys membrane

DNA Synthesis

Quinolones

Target:
DNA gyrase

Examples:

• Ciprofloxacin

• Nalidixic acid

• Sulfa Drugs

  Block:
  Folic acid synthesis

  Compete with PABA

RNA Synthesis

Rifampin

Blocks bacterial RNA polymerase

Selective

Actinomycin D

Binds DNA directly

Not selective

Toxic

Protein Synthesis

30S Subunit

Streptomycin

Binds:
16S rRNA

Causes misreading

Tetracycline

Blocks tRNA binding

50S Subunit

Erythromycin

Blocks translocation

Chloramphenicol

Blocks peptidyl transferase

Clindamycin

Same ribosomal site as chloramphenicol

Antivirals

Amantadine

Blocks viral uncoating

Tamiflu (Oseltamivir)

Blocks neuraminidase

Prevents viral release

Acyclovir

DNA chain terminator

Targets viral DNA replication