Microbiology 2 Exam

Positive Supercoiling

Coiling of DNA that increases the number of base pairs per turn.

Negative Supercoiling

Coiling of DNA that decreases the number of base pairs per turn.

Replicon

portion of the genome that contains an origin and is replicated as a unit.

Replisomes

A complex of 12 proteins involved in replication

Clamp Loader Complex

A protein that holds DNA Pol at the DNA strand during DNA replication

Tau

Binds and organizes E.coli replication through distinct domains

\# of E.coli DNA Pol

5, Pol III plays the largest role

Bacterial DNA Replication Steps

1. DnaA binds oriC, bending and separating the strands
2. DnaB separates strands and SSB attach
3. Primase synthesizes RNA Primer
4. lagging + Leading synthesized
5. DNA Pol I removes primers and replaces with DNA
6. DNA Ligase joins Okazaki fragments (3’ OH of growing strand and 5’ P of fragment)

Catenanes

Two loops of DNA linked when topoisomerases break and rejoin DNA to ease supercoiling

TTGACA

\+35bp, recognized by sigma factor

TATAAT

\+10bp, where DNA starts to separate

tRNA

70-95nt RNA folded into an L shape that recognizes mRNA codons with anticodons

Aminoacyl-tRNA synthetases

Catalyze AA attachment to tRNA. at least 20 exist for each amino acid.

16S rRNA

Binds SD sequence and 3’ RNA end

23S rRNA

Ribozyme that catalyzes peptide bond formation

t-RNA-fMet

Bacterial initiator tRNA that adds Met at AUG downstream of SD sequence. Commonly removed post-translationally

t-RNA-Met

Used by archaea and Euk, installs Met at elongating Aug codons using the 5’ cap as a reference.

Sec system

General secretion pathway. Moves unfolded proteins across the plasma membrane

Tat system

secretes only folded proteins

Two-step secretion

Type II, V, IX

One-step secretion

Type I, III, IV, VI, VII

Leader Sequence

Bacterial sequence transcribed into mRNA but not translated into AA

SD Sequence

Important for translation initiation

“Leaky Operon”

Operons have a low basal level of transcription

Genes for lactose metabolism

lacZ (Beta-galactosidase), lacY (lactose permease), LacA (Beta-galactoside transacetylase) . Inhibited by lacI when there is no lactose.

How does the lac repressor bind?

nonspecifically. Slides along major grove to reach an operator site recognizable by helix-turn-helix. Bends DNA to block RNA pol from accessing promoter.

How many operators can lacI bind to?

3

How many genes code for enzymes to synthesize tryptophan?

5

What control does the ara operon experience?

Positive and negative transcriptional control by araC


1. Inactive when ara present
2. Active when ara absent

Attenuation

Early termination of transcription or translation caused by step-loop folding patterns in the long leader region of mRNA (control in trp Operon)

Riboswitches

A folding of mRNA leader sequence in response to a bound effector molecule that determines if transcription continues or terminates.

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A form of transcription attenuation

RNA thermometers

Regulate translation by folding 2\* structures in the leader sequence dependent on temperature

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Measure temperature based on differences in thermal stability of GC and AU base pairs

sRNA, ncRNA, Antisense RNA

May inhibit or enhance translation

Regulon

Genes/operons controlled by a common global regulatory protein

Two-component Signal Transduction

When exposed to a signal in the cytoplasm, a sensor kinase transfers phosphate to response regulator in the extracellular environment to elicit transcriptional changes.

Second Messengers

* cAMP - CRP/CAP
* ppGpp/pppGpp
* Cyclic dinucleotides (c-di

Catabolite Repression

Regulation of transcription by both repressors and activators

Diauxic growth

Preferential use of one material over another. Lag occurs when preferred substrate is exhausted, then growth resumes using the second substrate.

CAP/CRP

Catabolite activator protein. Active when cAMP bound and interacts with RNAP to stimulate transcription.

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ALL catabolite operons contain a CAP binding site

Stringent response

Bacterial response to stress conditions. AA biosynthesis genes are transcribed, tRNA/rRNA transcribed less.

How does Chemotaxis work in E.coli?

MCPs bind to chemical signals in the encironment and activates sensor kinase CheA which autophosphorylates before relaying the phosphate to CheY.

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if MCP is more methylated (as analyzed by CheR and B), and CheY is active, CW rotation = tumbling

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If MCP is not methylated and CheA/Y are inactive, CCW rotation = running.

What regulation controls sporulation?

Phosphorelay, posttranslational protein modification, transcription factors, sigma factors.

* alt. sigma factors produce in response to starvation
* Phosphorelay begins with KinA when nutrient depletion detected
* Spo0A controls sporulation initiation

Quorum Sensing

Cell-cell communication mediated by signaling mols essential for virulence, symbiosis, biofilm production, and differentiation.

Restriction-modification

Innate system to restrict the growth of viruses.

Restriction endonucleases

Recognize and cut specific DNA sequences.

CRISPR/Cas System

Adaptive immunity


1. Adaptation: pieces of viral genome added to CRISPR array in surviving cell
2. Expression: CRISPR region transcribed, RNA matured by Cas proteins to create crRNAs
3. Interference: Cas-crRNAs block viral DNA/mRNA during infection

Condensates

Protein complexes that segregate and concentrate specific molecules to increase efficiency of biochemical reactions.

How many DNA polymerases function in Euk DNA replication?

at least 3

What are Telomerases important features?

* Internal RNA template (bps to G-tail)
* Reverse transcriptase activity (to maintain length of chromosome end)

How many RNA Polymerase do bacteria use?

1 - 5 subunits(polypeptides)

How many RNA Polymerases do eukaryotes have?

3

* RNAP I: rRNA
* RNAP II: mRNA
* RNAP III: tRNA and 5S rRNA

TATA Box

t absent for regulated genes. In eukaryotes, present at -31 to -26 for housekeeping genes.

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Transcription initiation by binding to TFIID.

What are the uses of 5’ cap?

Stabilizes and facilitates translation and splicing.

What are the uses of the 3’ Poly-A tail?

Aids in recognition, prevents degradation, and signals for transport

Is alternative splicing an option for bacteria and archaea?

No.

Translation in Bacteria

30S + 50S makes ribosome. Start codon binds 16S rRNA and initiator N-fMet-tRNA SD sequence. Termination may occur with or without Rho protein.

48S Complex

Forms when 43S binds to the bridged mRNA in eukaryotic translation initiation

80S ribosome

60S + 48S Subunit in eukaryotes

Vesicular transport

Moves proteins directly across a membrane in eukaryotes. Places proteins into ER, mitochondria, and chloroplasts

Twin-arginine translocation

Functions in chloroplasts and mitochondria

What does gene expression regulation look like in Archaea

* Rarely two-component regulatory systems
* TF can be positive or negative
* Histone-DNA complexes vary in size
* No post-translational modification

Apurinic site

A site in DNA where a base is missing (purine or pyrimidine). In the next replication round, one strand keeps the AP site and the other gets an incorrect random nucleotide.

Base analogs

Mutagens. Structurally similar to normal bases- mistakes when incorporated.

Intercalating agents

Mutagens that distort DNA to induce single nt pair insertions/deletions

Forward mutation

WT > Mutant

Reversion mutation

Mutant > WT

Suppressor mutation

WT phenotype restored by a second mutation at a different site than the original mutation.

Excision repair

Corrects damage that distorts the DNA double helix (base or nucleotide)

Recombinational Repair

Corrects DNA that has both bases of a pair missing or damaged with recombination with an undamaged chromosomal copy.

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Done by RecA.

SOS Response

Inducible repair system when damage is great.

* RecA destroys LexA to increase production of excision repair enzymes
* RecA initiates recombination repair >50 genes.

Fate of DNA in Horizontal Gene Transfer

* Integration (recombines)
* Separate (persists as a plasmid)
* Remains in cytoplasm (donor DNA can’t replicate
* Degradation (By CRISPR/Cas)

Homologous Recombination

RecA breaks DNA double stranded, reunion causes crossing over between long DNA regions with similar nt sequences

Site-specific Recombination

Recombination occurs by Recombinase at specific target sites in DNA mols.

Replicative transposition

Copy-and-paste

Simple transposition

Cut-and-paste

Rolling-Circle Replication

Unidirectional nucleic acid replication with polymerase and recombinase that rapidly synthesizes multiple copies of circular mols of DNA/RNA

Hfr Conjugation

F factor transfers itself in conjugation and drags the rest of the genome bridge, where process is often incomplete and connection breaks, breaking a portion of the copied genome into the F- cell.

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Resultant cell is still F-

Generalized Transduction

fragment of host same length as phage genome is mistakenly package and transfered in next infection, where it can be incorporated into the new host cell.

Specialized Transduction

Integration of phage takes place at a special attachment site in phage genomes called att sites.

Replica Plating

Screens for auxotrophic vs prototrophic parent based on ability to grow in the absence of a particular biosynthetic end product.

Conditional Mutations

Different phenotype under certain environmental conditions

Auxotrophic mutant

Unable to make an essential macromol (AA or nt)

MDA

Multiple Displacement Amplification

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Occurs at a single temperature and uses DNA pol from phi29 to synthesize new DNA

Metagenomics

Study of microbial genomes based on DNA extracted directly from the environment.

Each DNA fragment comes from a COLLECTION of genomes

Bioinformativs

Convert raw nt data into location and potential of genes

in silico analysis

locates genes in a genome map by identifying an ORF.

BLAST

base by base comparison of 2+ gene sequences, tentative function of gene or protein structure

Orthologues

Genes from different organisms with similar ORFs

Paralogues

2+ genes with similar nt sequences (duplication event)

Conserved hypothetical proteins

match known sequences in databases without understanding assigned function

Proteins of unknown function

Proteins of genes unique to an organism.

DNA Microarray Analysis

Determines what genes are expressed at a specific time.

RNA-Seq

Quantifies mRNA by measuring reads matching each gene. mRNA > cDNA, sequenced using NGS.

Metatranscriptomics

Describes transcriptome of an entire ecosystem

Proteome

Entire collection of proteins that an organisms produces.

Protein Modeling

Assumes proteins fold into a limited number of shapes and can be grouped into families. Determines 3D structure of proteins

Lipidomics, Glycomics, and Metabolomics

A cell’s lipid, carb, and small molecule metabolite profile

ChIP

Surveys protein DNA interactions in living cells.


1. Crosslinking
2. Sonication
3. Antibodies added and bound, + Sonication
4. Immunoprecipitation
5. Reverse cross linking
6. DNA Sequencing after proteins removed

Functional Genomics

Puts genomic information in a biological context

Systems Biology

Holistic study of cells for interactions.