MB 251 Exam 3

bacterial nomenclature rules

1. Genus is Capitalized Campybacter

2. species is lowercased jejuni

Gene/Protein Nomenclature

DNA/RNA: znuA; ftsz

Protein: ZnuA; FtsZ

DNA —> RNA —> Protein

Central Dogma

DNA makes RNA, RNA makes Protein

Horizontal Gene Transfer (Expression)

Recombination: Genetic info is used in a cell to make proteins needed for the cell to function. Involves Transcription and Translation leading to cell metabolizing and growing

Horizontal Gene Transfer (Recombination)

Genetic info transferred between cells in same generation

Vertical Gene Transfer

Bacteria a dividing and passing DNA to daughter cells

Chromosome

Main genetic element that main genetic element that is Essential for the cell's survival Tells cell how and when to do something

Gene

Genetic element in chromosomes that encode info to make specific RNA/Protein

1 Gene: 1000 Base Pairs

Genome

Entire complement of genes in cell including chromosomes and plasmids

Plasmids

Extrachromosomal DNA element that encodes Non-Essential genes; Expendable. Do not have to be transferred to daughter cells

Replicate separately from chromosomes, double stranded DNA, genetically mobile (can be passed from cell to cell), circular, and encode good genes like antibiotic resistance

• Can be lost and still survive

• Bacteria transformed with plasmids —> Conjugation/transformation

• Etopic (out of place) expression of non-native genes/proteins

• Ex. Lac promoter driving expression of GFP: +Lactose/-glucose (or lactose analogue) = +GFP = Green

• Vertical Transfer

Homologous Recombination:

Takes plasmid and integrates into chromosomes making it Essential; Genes guaranteed to transfer in every generation (daughter cells). --> Chromosomal genes

Griffith’s Experiment

• Smooth Strain: Capsulated, evades immune system, Virulent = Causes Diseases/death; Mouse Dies

• Rough Strain: No capsule, Non-virulent (Can’t cause diseases/death); Mouse Lives

• Heat Killed Smooth Strain: Heat treated bacteria. Mouse lives because cells are dead

• Rough Strain & Heat Killed Smooth Strain: Rough Strains mixed with heat killed smooth and Mouse Dies; Smooth Strains found out of mouse even though never added; Rough bacteria transformed into Smooth Bacteria

• Results in death: Live smooth, Live smooth + Dead smooth & Live rough + Dead smooth

Transformation

Cells have changed by a genetic change

Avery, Macleod, McCarty

Depleted sample of ONE component of smooth killed cells and assess transformation of rough cells. T2 Bacteriophage; Escherichia coli

4 Experiments: Only RNA removed, Only Protein removed, Only Lipids removed, Only DNA removed

1-3 Experiments RNA, Protease/Protein, Lipids Removed: Live Rough Bacteria —> Transformation into Smooth

4th Experiment DNA Removed: Rough cells stay Rough; Absence of DNA = Loss of Transformation

Does NOT DIRECTLY show that DNA is transformer of rough into smooth

Hershey Chase Experiment (Alfred and Martha)

Directly showed DNA = molecule transferred into cells allowing transformation. 

• Labeled Phage Proteins with Radioactive Sulfur → Incubation → Separation of phage and bacterial cells → Did not Observe Radioactive Sulfur/Proteins. Radioactive Sulfur = Proteins

• Labeled Phage DNA with Radioactive Phosphorus → Incubation → Separation of phage and bacterial cells → Observed Radioactive Phosphorous/DNA. Radioactive Phosphorous = DNA

Bacteriophage (Hershey Chase)

Viruses that specifically infect bacteria

• Protein coat and DNA gene

• Bind to surface of bacterial cell and inject genetic material into it

• Injected DNA incorporated into chromosomes —> more genes  that have competitive advantage ex. Capsule

DNA Replication (Semi-Conservative) Meselson-Stahl Experiment

Replicated in a Semiconservative Model; The Parental DNA both serves as templates for a copy to be made so any new DNA made is a hybrid

• Semiconservative Model (N15 Experiment): All Nitrogen in the cell’s DNA is N15. Next you switch to N14 and allow for Cell Division and all new Daughter cells will be a new N15/14 species. Eventually it will divide until you have almost all N14

After 20 minutes: Will be hybrids of the N15/14 and concentration goes down as time goes on

DNA Replication (Conservative) Meselson-Stahl Experiment

Parental DNA was a template but the same 2 strands from the OG parent were always passed down to a daughter cell and any copy of DNA would be completely new

• Conservative Model (N15 Experiment): Does not happen naturally; Double stranded DNA from parent is copied but never splits and forms a hybrid to turn into a new species

After 20 minutes: 50% will be Blue; 50% Red

Replication

• DNA makes DNA; DNA is copied; Make sure cell has all of the functions needed to survive

Starts at Origin of Replication (Ori)

Proceeds in both directions around chromosomes

1000 bp/second

New round starts before whole chromosome replicated

Theta Replication

Takes place in Cytoplasm (Nucleoid Region)

Transcription

• DNA makes RNA; DNA is converted to RNA

• RNA Polymerase carries this out

• Controlled/regulated process

  • Not all genes (DNA) are Always transcribed

  • Starts at +1 → Termination Sequence

  • Genes transcribed/translated into proteins when needed

  • Ex. Genes that code for proteins to metabolize lactose in DNA but lactose is NOT available, it is waste of time/energy 

  • o (sigma) recognizes promoter region and STARTS Transcription

Translation

• RNA makes Protein; RNA converted to Protein

• Synthesis of proteins from mRNA

• Not all RNA on transcript is translated

• Translation is from Start Codon (AUG) to Stop Codon (UGA/UAA/UAG)

DNA Replication (Starts with?)

Starts at Ori and goes in both directions around chromosome; Termination site is where two chromosomes separate

Central Dogma

Replication, Transcription, Translation

What unwinds DNA?

Gyrase and Helicase

Polymerase

Enzyme that makes nucleic acid strands from nucleic acid template

DNA Polymerase

 Makes new DNA from a DNA template during REPLICATION

RNA Polymerase

Makes new RNA from DNA template

• o (sigma) recognizes promoter region and starts Transcription

• During Transcription: Reads DNA Template strand 3’ - 5’. When MAKING RNA = 5’ - 3’ adding complement base

• EX. Reads 3’ - G - C - T - A 5’

• Adds COMPLEMENT 5’ - C - G - A - U 3’

• Recognizes and binds to -35/-10

Ori (Origin)

Start of Replication; Origin of Replication; 5 Prime and 3 Prime ends

Bacterial Chromosome Properties

Double stranded DNA, Circular, One chromosome per cell, Bi-directional Replication.

Bacterial Chromosomes

• Each line is a gene; coding region, 

• Outside circle is one Strand of DNA, Colors show different classes of genes

• Closed/Annotated Genomes: All genes included, Order on chromosomes, What their function is

Supercoiling

DNA supercoiled to fit into compact space of the cell

• Sometimes hides DNA binding sites

• Proteins required to locate specific locations on DNA

• Allows for regulating transcription/replication

Termination Site

Where two chromosomes separate

Promoter (Transcription PT. 1)

Far left region (upstream); DNA regulating expression (on/off) of a gene. RNA Polymerase = bonding site. Repressor/Activator binding sites

• Downstream = right; Upstream = left

Coding Sequence (Transcription PT. 2)

Middle; DNA template for producing RNA transcript

Coding Regions: Coding Strand: 5’ - 3’

Non-Coding Strand: 3’ - 5’

Nothing between geneA and geneB is transcribed

Terminator (Transcription PT 3.)

Far right region (Downstream);DNA sequence that tells RNA polymerase to stop transcription. Make sure cell does not waste energy transcribing DNA it does not need

Coding Strand (DNA Sequence)

5’ - 3’; DNA sequence is identical to transcribed RNA.

T in DNA is replaced by U in RNA

RNA Polymerase DOES NOT READ this strand

Non-Coding/Template Strand (RNA Sequence)

3’ - 5’; reverse complement of coding strand

Strand that RNA Polymerase READS when making RNA

Chargaff’s Rules (Transcription)

A = T

G = C

+1 Strand (Promoter Region)

Found through experiments only; START site of transcription. 1st nucleotide that RNA Polymerase Reads and Synthesizes.

1st base of NEW RNA

• -35/-10 are averages, 35/10 bases away from +1

RNA Polymerase (Subunits)

Holoenzyme: Core Enzyme: a2, B, B’ —> Synthesize RNA

o (sigma) Recognizes promoter region and STARTS Transcription

Transcription Initiation:

1. RNA Polymerase binds to -10/-35 region of the promoter. Sigma is the subunit that recognizes the sequence

2. RNA Polymerase moves down the DNA 5’ → 3’

3. RNA Polymerase synthesizes new mRNA at +1 site. +1 is the start of Transcription; 1st nucleotide transcribed into RNA

LoGo

Representation of Consensus sequence where more info is presented than traditional consensus sequence

• Size of the letter shows frequency among many sequences; Larger the letter more represented that nucleotide is at that position.

• Allows for all nucleotides to be viewed

Transcription Termination PT 1. (Rho-Independent)

Rho opens up, clamps to a C-rich site on mRNA and pulls mRNA through its central pore. When Rho catches paused polymerase, it keeps pulling mRNA separating it from the template and polymerase

Transcription Termination PT 1. (Rho-Dependent)

Stem-loop causes RNA polymerase to pause. A-U bps are weak, mRNA dissociates, and polymerase releases DNA

• mRNA transcript is  single stranded, allowing nucleotides to bp with complementary ones (A-U, G-C) on same strand of RNA

• A series of T’s is the genetic motif that provides evidence of Rho-Independent termination

Ribosome (Translation)

• Protein: RNA complex where amino acid subunits are linked together to form a protein. Site of protein synthesis

• Thousands per cell

• 2 Subunits: 30S and 50S in prokaryotes = 70S Complex (S=Svedberg units)

• Combination of rRNA and Protein

• Needed to transfer mRNA in Protein

• Binds at Shine-Dalgarno Site on mRNA Transcript

Genetic Code (Codon) (Translation)

Triple of nucleic acid bases (codon) that encodes a single amino acid

• Specific codons for Start/Stop

Anticodon (Translation)

• on tRNA recognizes codon on mRNA

• tRNA is amino acid carrying RNA

16S rRNA (Translation: Initiation)

Recognizes and binds Shine-Dalgarno sequence on mRNA, helping the ribosome start codon. Also good for structural stability

1. Initiation (Translation)

AUG = Codon. Incorporates amino acids; Arrival of new tRNA

P = Protein chain held here

2. Elongation (Translation)

Elongation = Exit of empty tRNA

3 stop codons: UAA, UAG, UGA

Termination (Translation)

Stop Codon = Uncharged tRNA will enter at A-Site

• Ribosome will Dissociate and mRNA and polypeptide will be released

Coupled Transcription & Translation

Translation can begin before transcription stops. 

• Bacteria don't have organelles which allows transcription and translation to happen in the cytoplasm

• mRNA transcripts do not need to be transported before translation starts

• Coupled transcription and translation is another mechanism by which bacteria can divide quickly

Most bacteria have how many chromosomes?

1 Chromosome and this DNA is CIRCULAR

Where does replication start and how does it move?

Bacteria initiate the process of replication at the origin (ori) and move in a bi-directional manner resemble the greek letter THETA

DNA Polymerase (High Fidelity Enzyme)

It doesn't make a lot of mistakes (less likley to result in mutations)

‘5 in-between 3’

-35, -10, +1, ATG, TGA, Terminator

Operon

A set of genes transcribed of the same promoter

Only in Bacteria; One promoter driving transcription of many genes

• All genes in operon transcribed at same time

• RNA Polymerase only binds once

• 1 Operon transcribed:1 mRNA produced

Frame Shift

Ribosome reads the incorrect 3 nucleotide code, incorporating the wrong amino acid in the polypeptide

Correct: THE CAT ATE THE RAT

Incorrect: ATH ECA TAT ETH ERA

Why Regulate Transcription and Translation?

It is expensive to make proteins

• Protein synthesis: 2 million ATP/Second

Transcriptional Regulation

Regulating how much transcript mRNA is produced

• Can be turned on/off/dimmed/high

When it can be turned on

• DNA = Promoter region

• DNA Binding Proteins: Regulators of Transcription; Activators/Repressors. Bind to DNA at Promoter regions

• RNA Polymerase: Enzyme transcribes DNA into mRNA

DNA Binding Proteins

• Regulators of Transcription; Activators/Repressors. Bind to DNA at Promoter regions

RNA Polymerase

• Enzyme transcribes DNA into mRNA

Translational Regulation

Regulating the translation of mRNA into protein; mRNA is made but not translated into protein

Post Translation Regulation

Regulating activity of synthesized protein; Protein is made but may/may not be active

Constitutive (Type of Transcriptional Regulation)

No regulation, gene is continuously transcribed, always on

Ex. rpoD

Repression/Negative Regulation (Type of Transcriptional Regulation)

STOP RNA Polymerase, Transcription Off

Ex. znuABC

Repressor Protein binds Promoter. Stops RNA Polymerase from starting transcription

NO RNA Polymerase binding, No Transcription = No mRNA = No Proteins

Activation/Positive Regulation (Type of Transcriptional Regulation)

Recruit RNA Polymerase, Transcription ON/Activated

Ex. luxCDABE

Activator protein binds upstream of promoter region

Recruits RNA Polymerase to bind/start Transcription

When would transcription of znuABC be on?

Low levels of zinc in the cell

When would transcription of znuABC be off?

High levels of zinc in the cell. Transcription off

Allosteric Regulation

•  Molecule binds to protein at a site other than active site causing the protein to change shape and activity

Quorum Sensing

Regulation of gene expression as a response to cell population density changes

• Low cell Density: Individual 

• High cell Density: Group

Bioluminescence

Light production, luxCDABE operon. Makes all required enzymes/substrates to produce light. Ex. Firefly light

• Low Cell Density, Low AI Signals = No Bioluminescence

• High Cell Density, High AI Signals = Bioluminescence. LuxR sense increase in AI signal and activates luxCDABE → Bioluminescence

Vibrio fischeri

Gram negative Bacteria, Naturally Bioluminescent, forms a symbiotic relationship with Hawaiian Bobtail Squid

Bioluminescence and Quorum Sensing

LuxR = Activator

• Light: Only binds DNA and activates when bound to AI signal molecule

• AI signal High = Binds to LuxR → Activation of Transcription → Bioluminescence

• No Light: Low AI signal will not bind with LuxR

• LuxR does not bind to promoter → No Transcription = No light

Hawaiian Bobtail Squid

Morning: Low V. fisheri = No light Production. Bacteria grows → Produce low AI signals → No light

Night: Population reached → AI levels High → Activate luxCDABE → Light Produced

Morning Again: Vibrios expelled from light organ → Low level bacteria → No light

Under which condition does the activator LuxR bind to the promoter of luxCDABE activating transcription?

High Cell Density/High AI molecules

LuxR protein is bound to promoter region of IuxCDABE operon when

High Cell Density

The Zur protein recognizes specific sequences in the promoter region of the znuABC operon and will only bind at this location, it will not bind the promoter region of the luxCDABE operon. 

lac Operon

One Promoter driving expression of lacZ, lacY, and lacA genes

• Creates Polycitronic genes

• Each frame translated into individual proteins: LacZ, LacY, LacA

• Codes for proteins that USE lactose

LacI

Used in: Repression & De-Repression, Constitutive Expression, Allosteric/Post-Translational Regulation.

DNA binding protein that senses lactose (Allolactase).Transcription is constituitive = always on.

When not bound, binds to promoter region of lacZYA operon called the Operator (O)

Repression when lactose is not present

Depression when lactose is present

Lactose is present: Not able to bind DNA and does not bound operator region (Depression)

CAP-cAMP

Used in Activation

(Jacob and Monod)

E. coli grown on two carbon sources show diauxic growth; Multiple growth phases in 1 growth experiment

lacZ: Lactose Hydrolysis

Cleaves Lactose into D-galactose and D-glucose

lacY: Lactose Transport

Driven by energy in the proton motive force. Transporter that brings sugar into the cell

lacZYA operon

Should be transcribed when Lactose is present

Model System Lag Phase

ALL GLUCOSE BEFORE LACTOSE! Then short period of stopping before Lactose consumed and growth starts again

Glucose

Monosaccharides. After transport used for Glycolysis

Lactose

Disaccharide. After transport enzyme breaks apart 2 sugars which requires input of energy

Promoter

-10/-35 RNA Polymerase bonding site

Operator

Binding site for lactose sensor/repressor (LacI)

CAP

Binding site for Catabolic Active Protein

Activator of lac operon

Only Activates when there is NOOO Glucose

Knows glucose is present through cAMP

Pi

Promoter for lacI gene

LacI repression

No Lactose, No transcription, LacI bonds to operator, Represson

LacI derepression

LacI does not bind to Operator, Transcription

cAMP

Only produced by E.Coli

Activated when No Glucose; tells cell about energy source

cAMP absent = glucose

cAMP present = no glucose

CAP-cAMP

Bonds to promoter region = CAP

recruits RNA polymerase to transcribe operon

Only binds to lac promoter when bound by cAMP

No Lactose/No Glucose

Transcription is off

LacI cAMP present/bound

Repression

No Lactose/ + Glucose

Transcription off

Repression

No cAMP/No LacI

No Activstor

Lactose/Glucose

Transcription is on, but the levels are LOW as glucose is present and there is no cAMP being made to bind CAP and activate transcription of the operon.

Derepression

Lactose/No Glucose

Transcription is on, and transcription is HIGH/ACTIVATED as glucose is gone, resulting in cAMP being produced, binding to CAP, activating transcription.

Derepression