Mircobiology Exam #3

Most sigma factors are highly conserved and help RNA polymerases identify the beginning of genes. They accomplish this by binding to which of the following sites on promoters?

-10 and -35 sites

icosahedral capsids

20 triangular sides Each triangle made up of at least 3 identical capsid proteins Allows small protein to cover a large volume Many animal viruses

Synonymous codons (those that code for the same amino acid) typically only differ in the base at position

3

What is the correct temporal sequence for factors binding to an mRNA in bacterial translation initiation?

30S ribosomal subunit, N-formylmethionine-tRNA, 50S subunit

If a segment of RNA reads 5′ GCCUUAA 3′, then the corresponding DNA template strand reads

5′ TTAAGGC 3′

inversion

A DNA sequence is flipped in orientation

rho independent termination

A GC rich region followed by a series of U residues GC residues form a stem loop structure. RNA polymerase binds the stem loop structure causing a pause in transcription The U's of the nascent RNA paired with A's of the DNA are not stable enough to hold the RNA polymerase on the template during the pause mRNA dissociates off DNA, polymerase released

bacteriophage

A bacteriophage or phage is a virus that infects Bacteria.

mutation

A heritable change in genotype (DNA sequence) that can lead to a change in phenotype (observable properties of an organism).

Wild-type cells of Escherichia coli can utilize xylose as a sole carbon source. To do so, they need to express the three genes of the xylAB operon (xylA, xylB) whose products encode proteins that modify xylose for catabolism. These genes are expressed at high level only when xylose is present in the environment. When glucose is present, however, expression of the xylAB operon is greatly reduced even when xylose is also available. Deletion of a gene called xylR in a different region on the chromosome causes the xylAB operon to be expressed at high level when cells are growing in a medium that contains any one of many sugars other than glucose.What is the function for the product of the xylR gene in regulation of the xylAB operon?

A negative regulator of the xylAB operon

reversion

A return to the normal phenotype. Mutant organisms that return to wildtype phenotype are called revertants.

mutant

A strain of any cell or virus differing from parental strain (mother cell or progenitor virus) in genotype.

conjugation F plasmid

A well-studied example in E. coli is the fertility factor (F factor). F contains: An oriT and tra genes. It is referred to as self transmissable. An oriR and replication maintenance genes. Multiple Insertion Elements and a Transposon that allow the plasmid to integrate into the chromosome. Conjugation begins with contact between the donor cell, called the F+ cell, and a recipient F- cell. Note that only a single strand of F DNA is transferred. The donor copies F by rolling circle replication displacing the strand transferring. The recipient synthesizes the complementary strand of the transferred strand

Which of the following codons could be used to signify the start of translation?

AUG or UUG CUG GUG

response regulator

Activated via phosphorylation by Sensor Kinase. Mediates "the response" by altering gene transcription.

how does CRP respond to the presence of cAMP

Acts as activator only when bound to cAMP High glucose → low cAMP levels → CRP inactive Does not bind operons → low level of lac transcription

osmolarity

All cells, including bacteria, have to maintain their internal solute concentrations and maintain the integrity of their membranes

Eukaryotes cap their mRNA at both the 5' (Methyl Guanosine Cap) and 3' (Poly Adenosine Tail)

Allows a eukaryotic to assess whether an mRNA is intact before export from the nucleus and translation by the ribosome.

envelopes

Allows fusion to host cell, organelle membrane Only occurs if host cell is not covered by cell wall Bacteriophages are NON-enveloped Envelope lipids come from host Not encoded by viral genome Proteins embedded in envelope may be encoded by virus Coats viral capsid as virus leaves cell or organelle

two component system

Almost 50 different two-component systems in E. coli Examples include phosphate assimilation, nitrogen metabolism, and osmotic pressure response. Sporulation in Bacillus sp. and Clostridium sp. are controlled by Some signal transduction systems have multiple regulatory elements

basic structural components of a virus

Always have a small genome May be: DNA or RNA, single or double stranded sense or antisense Always have a protein coat that protects the genome Capsid, made of capsomer protein.

auxotroph

An organism that is unable to synthesize a particular organic compound required for its growth.

genome regulation

An organism's genome encodes thousands of different proteins needed to handle many types of environmental contingencies.

plaques

Analogous to the bacterial colony; one plaque is the result of one virus infecting.

arginine operon

Arginine is a required amino acid for protein production. If the cell is in an environment low in arginine, the arginine biosynthesis operon will be expressed. The proteins encoded by the genes in this operon are enzymes that allow for the synthesis of arginine. If the cell is in an environment high in arginine, the arginine biosynthesis operon will be Repressed. Arginine acts as a co-repressor, binds the ArgR protein, allowing ArgR to bind the operator of the arg operon and block transcription.

assembly and release of lambda

Assembly is similar to that observe with T4. Capsid proteins self assemble into viral particles. Viral DNA is packaged in "head-full" manner. Tails are assembled to full heads. Release is different from T4 Makes protein to depolymerize peptidoglycan Bursts host cell to release progeny phage

maturation

Assembly of viral particle (assembling the capsid around the genome, incorporating any other required protein.

absorption

Attachment of the virus to the host cell through specific interaction.

response to nutrient availability

Bacteria also regulate gene expression that is necessary for building macromolecules.

polycistronic nature of the bacterial mRNA

Bacterial ribosome can readily assemble on start codons in the interior of the mRNA

chemical mutagens

Base analogs are incorporated in place of ATCG. Base modifiers modify ATCG usually via oxidation Intercalating Agents alter DNA structure by inserting itself between bases

initiation

Bind polymerizing machine, first monomer to template DNA polymerase, RNA polymerase, ribosome

rifampin/rifampicin

Bind the β subunit of RNA polymerase inhibiting polymerase activity. Bacteriostatic Not often used on its own.

negative regulator/repressor

Bind to regulatory sequences in the DNA and prevent transcription of target genes. Most often repressors block sigma factor of RNA polymerase from binding promoter. Bind DNA at sequence called operator sequence Repressors can require co-factors (ligand) to repress or be released from binding site. Cofactors required for repressor binding are called co-repressors. Cofactors that relieve repression are called inducer

positive regulator/ activator

Bind to regulatory sequences in the DNA and promote transcription of target genes. Often acts to recruit or stabilize sigma factor interaction with a promoter. Binding site generally upstream of -35, -10 sequence. Called activator binding site. Interacts with alpha subunit RNA polymerase. Can also alter DNA structure to allow binding of sigma factor. Activators can require co-factors to bind DNA. ALL REFERRED TO AS INDUCERS

Which sentence correctly describes inducers and co-repressors?

Both interact with a cognate repressor to regulate transcription of an operon.

establishing lysogeny

CI represses transcription of all λ genes, except CI. Helps maintain CI repressor concentration. λ Integrase (Int) and Integration Host Factor (IHF) recognize the attP sequence on phage DNA and attB sequence on the bacterial chromosome and integrates λ genome in to the bacteria chromosome . After integration λ is now termed a prophage.

generalized transduction

Can transfer any gene from a donor to a recipient cell. Results from the lytic growth of a bacteriophage Infection of host strain with a lytic bacteriophage. Examples: P1, P22, & T4 Viral head can package only a limited amount of genetic material. "Dead End" Infection: Phage that packages host genomic material instead of the phage genome can only infect once! Often used as a molecular tool to alter the genotype of bacteria in the laboratory. Infect host that has trait you want (like antibiotic resistance) Harvest phage Infect new host Select for trati in the new host.

specialized transduction

Can transfer only a few closely linked genes between cells. Results from lysogeny of bacteriophage. temperate phage only The result of aberrant excision of prophage from the host chromosome. Results in a hybrid phage-host genome being packaged. Can only transfer genes immediately adjacent to the phage attachment site.

nonsense mutation

Changes the amino acid sequence to a stop codon

missense mutation

Changes the amino acid sequence to another

frame shift mutation

Changes the open-reading frame of the gene

CRP

Concentrations of cAMP are assessed via cAMP Receptor Protein CRP is an activator protein Binds next to promoter, stimulates open complex Increases transcription of lac and other operons

post translational regulation

Controls the activity of preexisting enzymes. Typically a protein is modified to change its activity. Very rapid process (seconds)

regulation of gene expression

Controls the amount of an enzyme Gene expression: transcription of gene into mRNA followed by translation of mRNA into protein. Generally regulation occurs at the level of transcription by controlling the amount of mRNA. Sometimes regulation can occur at the translation level by controlling whether an mRNA is translated. Slower process (minutes)

DNA retro

DNA genome transcribed by host RNA pol to RNA intermediate. viral reverse transcriptase makes genomic DNA

viral genomes

DNA or RNA Single (ss) or double-stranded (ds) Linear or circular RNA genomes are either sense (+) or antisense (-) Includes genes encoding viral proteins Capsid Envelope proteins if virus is enveloped Any polymerase not found in host cell

horizontal gene transfer

DNA transfer between organisms that is not due to bacterial reproduction. Horizontal DNA transfer can change the genotype and phenotype of a cell

Which of the following is the enzyme complex that catalyzes transcription in bacterial cells?

DNA-dependent RNA polymerase

silent mutation

Does not change the amino acid sequence

Conjugation requires

Donor Cell to make contact with the Recipient Cell. If transfer require contact between a donor and a recipient cell, than conjugation has occurred.

promoter recognition by sigma factors

Each sigma factor recognizes a specific consensus sequence. Sequence in the promoter Sequences are generally at the -35bp and -10bp upstream of the transcription start site. Mutations in these sequences can cause changes in transcription levels.

physical mutagens

Either ultraviolet radiation or ionizing radiation.

DNA repair is divided into two types:

Error-proof repair pathways Error-prone repair pathways

excision repair

Excision repair enzymes remove a section of the DNA that has been damaged (by radiation or mutagen). DNA polymerase I fills the gap. DNA ligase links the new DNA with the old.

generation of F'

F can leave the chromosome via recombination. Depending on where recombination occurs, F may be altered, missing and/or gaining new DNA. Referred to as F' If F' is missing vital functions, called F' defective. If it carries a new gene, F' gene name (ex F'met) Will transfer if oriT is intact Will be maintained if oriR is intact.

mutation rates

For most microorganisms, mutations occur at a frequency of 10−9 per base pair per replication event. The proof-reading activity of DNA polymerase give in an error rate of 10-6-10-7 DNA Repair systems reduce the rate of mutation to 10-9 DNA viruses have mutation rates 10-100× greater. The mutation rate in RNA genomes is larger than DNA genomes. Some RNA polymerases have proofreading capabilities However, RNA repair mechanisms similar to DNA repair mechanisms do not exist

prophage induction

For λ prophage to become lytic, CI repressor must be inactivated. This occurs under conditions of DNA damage DNA damage activates the bacterial protein RecA. RecA binds CI and stimulates autocleavage of the protein. Transcription of phage genes occurs. Int and Excission protein (XIS) act to recombine λ genome out of the chromosome.

The energy used directly for the elongation step of translation comes from

GTP hydrolysis

catabolite repression

Glucose is easiest sugar to digest (catabolize) If glucose is present, lac operon not transcribed. Glucose is utilized first. The biphasic curve of a culture growing on two carbon sources is often called diauxic growth. A catabolite (glucose) is repressing the expression of operon.

inducer exclusion

Glucose transport by the phosphotransferase system causes catabolite repression by inhibiting the LacY permease activity.

splicing strategy

HIV

Cells possessing an integrated F plasmid are called

Hfr (high frequency of recombination) High rates of genetic recombination between genes on the donor chromosome and those of the recipient

error proof repair

High fidelity repair mechanisms for non-catastrophic damage. includes direct reversal and repair of single stand damage

the Baltimore virus classification

In 1971, David Baltimore proposed that the classes of viruses be distinguished by two main criteria. Genome composition (RNA or DNA) The route used to express messenger RNA (mRNA) Baltimore shared the 1975 Nobel Prize in Physiology or Medicine for discovering how tumor viruses cause cancer.

how quorum sensing and autoinducers can affect gene expression

Induction of a quorum-sensing gene system requires the accumulation of autoinducer. At a certain extracellular concentration, the secreted autoinducer interacts with a bacterial signal receptor. This signal transmitted to a response regulator which alters the genes expressed. Quorum Sensing was originally discovered when examining the symbiotic relationship of the Bobtail Squid with Vibrio fischeri. Responsible for the light produced by squids light organ.

temperate bacteriophage

Infection can be virulent or can lead to to maintenance of the virus within the cell in a dormant state, called lysogeny (example Lambda).

Pseudotemperate Bacteriophage

Infection establishes a permanent relationship with the host. Virus is produced and secreted continuously without host cell lysis (example: SP10).

assembly and release

Infectious viral particle is assembled in the cytoplasm of the host. Release is different for Naked (lyse cell) and Enveloped Virus (bud/shed from cell).

anti sigma factor

Inhibit by binding specific sigma factors and blocking their access to the RNA polymerase. The release of sigma factors from their anti-sigma factor proteins is important for flagella construction.

overview of translation of RNA to protein

Initiation factors bind ribosome to the ribosome binding site, known as the Shine Delgarno Sequence (AGGAGG in E. coli) 1 Translation begins at the AUG start codon. Elongation Polymerization, movement of ribosome along mRNA EF-Ts, EF-Tu, EF-G bring GTP energy Termination Releasing factors undock ribosome from mRNA

What is the role of the first structural gene (lacZ) in the lac operon? Select all that apply.

It isomerizes lactose into allolactose. It breaks down lactose into glucose and galactose.

lacI

Lac repressor, protein blocks transcription of the lac operon.

absence of lactose

LacI binds as a tetramer to the operator region. It represses the lac operon by preventing open complex formation by RNA polymerase.

Proteins in prokaryotes are designated by capitalizing the first letter of the name and NOT using italics.

LacZ - refers to the protein encoded by the lacZ gene.

lacY

Lactose permease, membrane protein that imports lactose from the extracellular environment.

lambda life cycle overview

Lambda Absorbs to the LamB porin of E coli. After injecting its genome it can take on one of two pathways lytic or lysogenic

filamentous/helical capsid

Long tube of protein, with genome inside Tube made up of hundreds of identical protein subunits arranged in a helical symmetry. Tube length reflects size of viral genome Some viruses with helical capsids have multiple genome segments.

error prone repair

Low fidelity repair mechanisms for catastrophic DNA damage repair of double strand break damage, a break in the DNA

examples of systems regulated by quorum sensing

Luminescence Virulence factors Switching from free-living to growing as a biofilm There is some evidence to suggest that bacteria can even sense signals that are not their own!

phage lambda makes a critical decision

Lytic vs Lysogenic Switch made up of 3 proteins: CI, CII, CIII. CI represses λ phage gene expression. CII and CIII assess cell nutrition. Phage assesses Host cell nutrition via CII and CIII. CII positive regulator and promotes expression of CI (repressor). CIII is an inhibitor of host cell protease that cleaves CII, but is only effective against low levels of protease.

release

Mature Virions are released from the host cell via lysis (naked virus) or budding (enveloped virus).

Activation of the lac Operon by cAMP-CRP

Maximum expression of the lac operon requires the presence of cAMP and cAMP receptor protein (CRP). The cAMP-CRP complex binds to the promoter. Interacts with RNA pol to increase the rate of transcription initiation

genome replication

Mechanism dependent on the type of nucleic acid the virus uses for its genome.

mismatch repair

Mismatch Repair enzyme removes the un-methylated new base of the DNA. DNA polymerase I fills the gap. DNA ligase links the new DNA with the old.

complex capsids

Mixture of icosahedral and filamentous symmetry Many bacteriophages (virus that infects bacteria)

temperate phage (lambda)

Model phage for understanding lytic vs lysogenic switch. dsDNA genome Linear, with complimentary single stranded ends. Circularizes upon cell entry. Complex protein structure surrounds genome Called a capsid with "complex symmetry."

point mutations

Mutations that change only one base pair Can lead to single amino acid change in a protein, an incomplete protein, or no change at all

selectable mutations

Mutations that give the mutant a growth advantage under certain conditions. Useful in genetic research

induced mutations

Mutations that occur due to agents in the environment or through deliberate manipulation by humans.

non selectable mutations

Mutations that usually have neither an advantage nor a disadvantage over the parent. Detecting such mutations requires examining a large number of colonies and looking for differences (screening).

lytic pathway

New virions are made, the cell lyses releasing virions. Phage quickly replicates, kills host cell Temperate phage can choose to be lytic or lysogenic Generally lytic when host cell conditions are good or when conditions are very bad (e.g., cell damaged, large scale DNA damage, etc)

most mutations are silent

No effect on organism Mutations in regions between genes Mutations that change 3rd base of a codon Mutations that change 1 amino acid into a similar one Protein still retains normal function Mutations that change a protein that is not needed for growth in current conditions.

what is a virus

Non-cellular infectious particle. Requires a host cell to replicate its genome. Requires host cell metabolism for particle construction. Living thing?

T4 assembly and release

Packaging motor is assembled Double-stranded DNA is pumped into head under pressure using ATP T4 can package DNA of a certain number of base pairs. It CANNOT tell whether the it is packaging its own DNA or host DNA of the same size. We will return to this when we talk about Transduction. After head is filled with DNA, T4 tail, tail fibers, and other components are added. After Assembly of enough virions the cell will lyse due to osmotic shock. Typically T4 has a burst size of 100 virions.

vision assembly

Packaging of the Viral Nucleic Acid into capsid protein. Some viruses also package viral enzymes/accessory proteins.

β-galactosidase converts lactose to glucose

People also make β-galactosidase If not, person is lactose-intolerant

transduction requires

Phage are smaller than cells, but can still be filtered out of solutions/cultures.

lysogenic pathway

Phage genome integrates into host cell genome Replicates only when host genome divides Generally lysogenic in moderate cell conditions Phage can reactivate to become lytic, kill host Phage is quiescent Integrates into host cell genome Replicates only when host genome divides Generally lysogenic in poor to moderate cell conditions Phage can reactivate to become lytic, kill host

light repair

Photolyase excise thymine dimers. DNA polymerase I fills the gap. DNA ligase links the new DNA with the old.

Rho-dependent termination

Polymerase slows at pause site, GC-rich sequence, forms stem loop Rho (ρ) factor binds to mRNA Slides along mRNA to polymerase Dissociates polymerase, mRNA off of DNA

autoinducers

Quorum sensing bacteria produce and release chemical signal molecules that increase in concentration as a function of cell density. The detection of a minimal threshold stimulatory concentration of an autoinducer leads to an alteration in gene expression.

conjugation examples

R1 is a conjugal plasmid that codes for five different antibiotic resistances! The anthrax toxin genes of Bacillus anthracis are found on the plasmid pX01 which is a conjugal plasmid. Strain of Bacillus cereus a soil bacterium that causes mild food poisoning have been found with this plasmid!

elongation

Read template, add next monomer DNA, RNA, Protein

wide scale DNA damage activates

RecA

sigma factor

Recognizes promoter region of gene Released soon after the start of transcription. there can be multiple sigma factors, E. coli has 7

why is recombination important

Recombination is the primary way in which genomes of bacteria change. It can repair "broken" genes. It can add new genes to a genome. Homologous Recombination can actually change a non-pathogenic bacteria into a pathogenic bacteria!

wild type

Refers to an organism with the typical genotype found in nature.

termination

Release machine and completed product

uncoating

Removal of Capsid from the genome. mechanism is dependent on the specific virus

the mosaic nature of genomes

Result of heavy horizontal gene transfer, recombinations, and a variety of mutagenic and DNA repair strategies Genome changes are selected for through evolutionary process.

rRNA

Ribosomal RNA, synthesizes protein

the RNA binding sites in the ribosome

Ribosome subunits are separate when not synthesizing proteins Subunits join near the 5' end of mRNA to initiate synthesis of a protein Ribosome contains 4 binding sites for RNA: 1 for mRNA and A,P,E sites for tRNA's tRNA held at A & P sites only if its anticodon complements the mRNA codon Close association of A & P sites force base pairing with adjacent codons (maintenance of correct reading frame)

Heat shock response in E. coli

Secondary structures at the 5´-end of rpoH mRNA obscure access to ribosome-binding sites resulting in little RpoH being produced. The small amount of RpoH produced is bound by chaperones and degraded. Heat melts the secondary structure allowing access of the ribosome and translation of the of more rpoH mRNA. Moreover, heat draws chaperones away from the protein RpoH, allowing them to accumalte and bind RNA polymerase.

transcription initiation

Sigma factor binds core RNA polymerase Forms RNA polymerase holoenzyme RNA polymerase binds promoter Sequence on DNA 10 and 35 bases upstream of start site Polymerase unwinds DNA at promoter Open complex Transcription begins and the Sigma factor is released.

secondary messengers

Small molecules like cAMP that don't serve as biosynthetic precursors but instead have regulatory function change the activities of regulatory proteins and regulatory RNAs. They often are involved when environmental changes require complex physiological responses for survival.

strategies for viruses

Synthesis of a polyprotein that is subsequently cleaved to component proteins. Synthesis of separate mRNAs by using different promoters of splicing. Segmented genome

T4 life cycle

T4 absorbs to E. coli, through tail fiber binding of LPS. Tail pins make contact with the outer membrane of E. coli. Injection: The tail sheath contracts, pushing the the tail tube through the outer membrane. T4 Lysozyme makes a small hole in the peptidoglycan and the DNA is injected

T4 DNA replication

T4 has a dsDNA linear genome The genome is circularly permuted due to the replication scheme. A genome unit is replicated. Each genome unit is recombined to produce a long polymer of genome called a concatemer. This concatemer is then cleaved at different points by a viral endonuclease. Each genome equivalent will contain 1 copy of all of the necessary genes, plus extra copies of genes at each end. We call this a circular permutation

T4 genes and gene expression

T4 has a large LINEAR genome divided into genes that are transcribed at early, middle and late time points during infection. Early genes: Nucleases, Replication proteins, Sigma Factors, RNA polymerase modifying proteins Middle genes: RNA polymerase modifying proteins Late genes: Capsid proteins, Assembly proteins, Release proteins

temperate phage lambda

Temperate phage Model phage for understanding lytic vs lysogenic switch. dsDNA genome Linear, with complimentary single stranded ends. Circularizes upon cell entry. Complex protein structure surrounds genome Called a capsid with "complex symmetry."

tRNA has a very specific structure.

The Anticodon loop will base pair (interact) with the complimentary mRNA codon. This interaction is how the codons of mRNA are translated into an amino in a growing protein. Each tRNA, except those complimentary to the stop codon, is "charged" with an amino acid at the Acceptor End.

Proteins that bind DNA can alter transcription rates in 2 different ways:

The binding event can block transcription: negative regulation. The binding event can activate transcription: positive regulation.

dehydration synthesis

The chemical reaction facilitated by a Ribosome the bond linking the amino acids is called a peptide bond. Nascent proteins are often referred to as peptides or polypeptides.

osmotic pressure

The environment is also filled with solute, though it is at different concentration than This causes a pressure to be put on the cell membrane called

tRNA "charging" is carried out by a specific aminoacyl-tRNA synthetase.

The enzyme recognizes tRNA structure in the D-Loop and Anti-codon stem of the tRNA and links the acceptor end of the tRNA to the appropriate amino acid. Multiple aminoacyl tRNA synthetases are required to charge the 20 different amino acids to the appropriate tRNAs Fidelity of recognition process between tRNA and aminoacyl-tRNA synthetase is critical. Incorrect amino acid could result in a faulty or nonfunctioning protein

reading the code

The three possible reading frames in protein synthesis Sequence of nucleotides in an mRNA molecule is read from 5' to 3' end in sequential sets of 3 nucleotides The same RNA sequence could potentially specify 3 different AA sequences, depending on reading frame Only one of these reading frames contain the actual message! A signal at the beginning of each mRNA sets the correct reading frame.

Control of ratio of σ factors determines global control of protein synthesis

These can be controlled by altered transcription, translation, proteolysis, and anti-sigma factors.

How do anti-sigma factors function to regulate gene expression?

They bind reversibly to sigma factors to prevent their association with core RNA polymerase.

spontaneous mutations

Those that occur without external intervention Spontaneous mutations are generally the result of an errors made by DNA polymerase during base pairing.

microbes respond to changing environment

To survive adverse conditions, thrive in good conditions, compete for a niche, and/or move to a better niche response includes: Sensing the environment - lac operon Glucose and Lactose concentrations are sensed. Changing gene expression - lac operon Genes necessary for lactose are expressed at high levels when necessary.

lacA

Transacetylase, role is still under debate.

Coupling of transcription and translation

Transcription creates mRNA Multiple mRNAs made from a single gene Ribosomes bind mRNA While mRNA is still being created Multiple proteins made rapidly from each mRNA Advantage of not having a nucleus

conjugation

Transfer of DNA between bacteria via cell-cell contact. Referred to as"Bacterial sex" Can transfer plasmids and sometimes chromosome material (via Hfr). Transfer requires an origin of transfer (oriT) and mating apparatus (tra genes). Usually, but not always found on the same plasmid. Single strand of DNA is transferred! Strand replicated in the recipient during transfer.

Hfr strains and chromosome mobilization

Transfer proceeds from oriT through the chromosome. 100 minutes for transfer of the entire E. coli chromosome. Very rare! Most matings are "interrupted." Transferred DNA must recombine with recipient Chromosome to be maintained. Recipient Cells do NOT become F+ unless the ENTIRE chromosome is tranferred!

transformation

Transformation is the process of importing free DNA into bacterial cells. The cells need to be competent. Many cells are capable of natural transformation. Some Gram-positive bacteria transform DNA using a transformasome complex. Some Gram-negative bacteria transform DNA without the use of competence factors (CF). Others require artificial manipulations. Perturbing the membrane by chemical (CaCl2) or electrical (electroporation) methods. DNA is not always incorporated, quite often it is degraded.

EnvZ-OmpR system

Two-component system that helps bacteria maintain osmotic homeostasis. Inner-membrane histidine kinase EnvZ phosphorylates itself under changes in osmotic pressure. EnvZ-P binds OmpR then phosphorylates OmpR (transfer P). OmpR-P binds upstream of ompF operon. Activates transcription of ompF in cases of low osmolarity. Represses transcription of ompF in cases of high osmolarity. OmpR-P binds upstream of the ompC operon activating transcription in the case of high osmolarity.

ames test

Uses bacterial strain auxotrophic for histidine. Has frameshift mutation in hisG gene Cannot grow unless histidine is supplied Place on medium + chemical Mutagen causes reversion Changes mutation to normal form Rare mutation More colonies = stronger mutagen Most mutagens are carcinogens. The Ames assay can be modified to test for the mutagenicity of chemicals processed in the liver.

asymmetrical/ irregular capsids

Viral capsid proteins are arranged without symmetry. Often have irregular shape. Tend to be larger viruses Poxviruses

penetration

Viral entry into the cell via fusion of envelope to the plasma membrane, endocytosis, or direct injection (bacteriophages)

T4

Virulent bacteriophage that infects Escherichia coli. Has only a Lytic lifestyle (ie. it can only make copies of itself and kill the cell) Complex capsid symmetry. Linear, dsDNA genome. Tail fibers bind E. coli LPS during absorption.

plaque assay

Virus infects a lawn of host cells creating clear zones called plaques.

overview of the virus life cycle

Virus replication is typically characterized by a viral "growth" curve. Virus concentration drops when first introduced into culture. Coincides with absorption and penetration of virus.

Culturing, Detecting, and Counting Viruses

Viruses replicate only in certain types of cells Bacterial viruses are easiest to grow; model systems. Animal viruses (and some plant viruses) can be cultivated in tissue or cell cultures. Plant viruses typically are most difficult because study often requires growth of whole plant.

phage lambda attachment and injection

Viruses take advantage of Bacterial cell receptors, pores, and structures. λ attaches at LamB, a porin involved in maltose transport. Utilizes the cells Mannose transporter system facilitate entry into the cytoplasm.

electromagnetic radiation

X-rays and gamma rays: Break the DNA Ultraviolet rays: Form Thymine dimers

Escherichia coli deleted for the xylR gene (lack the gene entirely) will not express the xylAB operon (xylA, xylB) at high levels in growth medium rich in both glucose and xylose. Pick the hypothesis below you believe best explains this observation.

Xylose is an inducer that induces transcription of the xylAB operon. In the presence of the glucose, very little xylose is imported into the cell due to inducer exclusion. This results in low transcription of the xylAB operon.

F plasmid is an episome

a genetic element that can integrate into host chromosome.

An inducer causes gene expression by binding

a repressor protein to remove it from the DNA.

insertion

addition of one or more bases

intercalating agent

alter DNA structure by inserting itself between bases

phage conversion

alteration of the phenotype of a host cell by a lysogenization Nondefective temperate phage lysogenizes a cell and becomes a prophage Host cell becomes immune to further infection by same phage Other phenotypic changes can also occur

Most tRNAs become charged with

an amino acid

auxotrophy

an organism cannot synthesize an organic compound required for growth.

constitutive proteins

are needed at the same level all the time Microbial genomes encode many proteins that are NOT needed all of the time.

PlacI and PlacZYA

are the promoters of each operon. Please note this operon produces a polycistronic message.

Aminoglycoside Antibiotics like Kanamycin, Gentamycin, Streptomycin (initiation)

are thought to bind the initiation complex (30s-mRNA-tRNA) and block construction of the complete ribosome.

transformation requires

bacteria to be competent and to take up naked DNA. Enzymes (DNAse) in solution may destroy DNA before it is able to be taken up. Mutants in Homologous Recombination are not capable of transformation.

Quinolones

bind Topo IV and DNA gyrase inhibiting their activity Broad Spectrum, Bactericidal Examples: Ciprofloxacin and Levofloxacin

Anti-anti-sigma factors

bind anti-sigma factors, releasing sigma factors to activate transcription.

Sigma factors are responsible for

binding of RNA polymerase to consensus sequences on DNA.

Various sigma factors play a role in gene expression by

binding to a variety of different -10 and -35 sequences.

Tetracycline (elongation)

binds the 30s subunit and inhibits binding of amino-acyl tRNA to the A-site of the ribosome.

Chloramphenicol (elongation)

binds the 50s subunit and inhibits the peptidyltransferase activity.

Increased synthesis of _____________ is a direct result of decreasing glucose levels.

cAMP

rich growth media

cAMP low, higher level of protease, less CII. CI repressor is not made and lytic cycle favored.

The AraC-like regulators differ from the LacI repressor in that only the AraC-like regulators

can activate transcription by direct interactions with RNA polymerase.

mutagenic agents

chemical agents and radiation and sometimes even infectious agents.

RecA

cleaves the LexA repressor protein This allow expression of: SulA which inhibits FtsZ and the division of the cell. UvrA an enzyme part of the excision repair pathway DNA Polymerase V which is an error prone polymerase that can synthesize DNA rapidly across DNA lesions. This allows DNA polymerase III to finish replicating the chromosome.

codon

codes for an amino acid. Note the start (AUG) and stop codons (UAA, UAG, UGA) Note the degenerate nature of the codons due to the third base position. Called the "wobble" position

virion

complete virus particle

A DNA site that binds a regulatory protein can be indicated when the DNA

contains an inverted repeat

In E. coli, eight enzymatic steps are involved in the biosynthesis of arginine from glutamate. The synthesis of the required enzymes is subject to control by the ArgD protein, which, on its own, binds weakly to a regulatory sequence just downstream of the promoter regions for these genes. Upon the addition of excess arginine to the growth medium, the respective mRNA molecules are no longer produced. What is the likely role of arginine in the control of these genes?

corepressor

Presence of glucose affects signal inside cell called

cyclic adenosine monophosphate (cAMP). Entry of glucose lowers the activity of adenylate cyclase, the enzyme that makes cAMP.

repair of single strand damage

damaged DNA is removed and repaired using opposite strand as template

when arabinose is present

each AraC subunit binds to an arabinose molecule and assumes a more compact shape. Activates gene expression by recruiting RNA polymerase to the promoter.

what are the two steps of the latent period

eclipse maturation

A single base-pair mutation occurs in a promoter sequence of a gene. As a result of the mutation, what are the possible effects on the transcription level of the gene?

elimination increase reduction no effect

polycistronic

encode several different proteins translated from same mRNA molecule

True or False: While bacteriophage are ubiquitous in the environment, the gut microbiome doesn't contain bacteriophage.

false

burst size

following the latent period the virus is released number of virions released Burst Size is dependent on the type of virus. Some viruses are able to generate larger number of virions than others. Viruses the lyse the cell will be released all at once. Viruses the bud/shed (envleoped viruses) may release over a longer time period. The end result of viral infection in many cases is cell death.

secondary structure

found in most proteins, consists of coils and folds in the polypeptide chain. Hydrogen bonding of the polypeptide backbone.

LATE phage lambda gene expression

gene expression - initiates from PR' (also called Plate) Requires the Q protein anti-terminator. Late genes encode phage structural components and lysis enzymes.

enveloped viruses

have an outer lipid bilayer membrane containing various proteins.

poly protein strategy

hep C

poor growth media

high cAMP, low level host cell protease that degrades CII. CIII inhibits remaining protease CII promotes expression of CI repressor. CI repressor prevents further λ gene expression and lysogeny is favored.

(+/-)ssDNA

host DNA polymerase

dsDNA

host or viral DNA polymerase

sensor kinase

in the cell membrane Binds to environmental signal Activates itself via phosphorylation

virulent bacteriophage

infection results in cell lysis and the production of many progeny phage (example T4).

segmented genome strategy

influenza

EARLY phage lambda gene expression

initiates at PL and PR and terminates after N and Cro. Leads to production of only N and Cro. N promotes expression of middle genes

MIDDLE phage lambda gene expression

initiates from PL and PR N protein, transcriptional anti-terminator. Leftward transcription proceeds through the int gene. Produces Int (integrase), Xis(excisionase), and regulatory protein CIII. Rightward transcription proceeds through the Q gene. Produces DNA replication proteins (O, P), and regulatory protein CII.

Sigma factors are necessary in which phase of transcription?

initiation

DNA binding proteins

interact with DNA in a sequence-specific manner Interactions are between amino acid side chains and chemical groups on the bases and sugar-phosphate backbone of DNA Major groove of DNA is the main site of protein binding Inverted repeats frequently are binding site for regulatory proteins Small molecules influence the binding of regulatory proteins to DNA

AraC

is a member of a family of regulatory proteins that have dual regulatory functions. controls the expression of proteins that allow microbes to metabolize the sugar arabinose. forms a dimer that can assume one of two conformations depending on whether arabinose is available. When arabinose is absent, represses gene expression, but when it is present, AraC activates these same genes.

guanosine tetraphosphate (ppGpp)

is a second messenger that allows cells to handle abrupt changes in nutrient availability. Allows switching from rapid growth to slower growth. Nutrient scarcity leaves many ribosomes idle. Idling ribosomes trigger the synthesis of ppGpp, ppGpp RNA polymerase and lowers its ability to transcribe ribosomal RNA (rRNA) genes. The response causes a decrease in rRNA transcripts made for ribosome assembly. This leads to fewer ribosomes and an overall decrease in growth.

RNA

is a transcription of the DNA code. Transcription of ATCG of DNA code to AUCG of RNA. The main RNAs found in a cell are mRNA, tRNA, and rRNA

tertiary structure

is determined by interactions among various side chains (R groups)

osmosis

is effectively the diffusion of fluid, through a partially permeable membrane, from an area of low solute concentration to an area of high solute concentration. Cells are essentially bags of fluid filled with solute, separated from their environment by the permeable plasma membrane

eclipse

is the period in which the virus copies its genome and produces proteins.

translation

is the process of translating mRNA sequence into amino sequence.

quorum sensing

is the regulation of gene expression in response to fluctuations in cell-population density.

lactose

is used for food Cannot pass through cell membrane Lactose permease allows entry PMF used to bring lactose inside cell Must be converted to glucose to be digested

lacO

lac operator, DNA sequence that binds LacI. DOES NOT PRODUCE PROTEIN !!!

Genes in prokaryotes are designated in Italics with the first letter NOT capitalized:

lacZ - refers to the gene or DNA sequence

bacterial RNA polymerase

large molecular machine 5 proteins in the core complex (holoenzyme). (2) α : interacts with regulatory proteins β : catalytic activity β′: involved binding and staying bound to DNA. ω: stabilizes the complex

Consider this image illustrating the various protein-DNA interactions that occur in the promoter region of the lac operon. The configuration shown in panel A would occur in cells with _________ levels of lactose and _________ levels of glucose.

low/high

what are the three types of RNA involved in translation

mRNA tRNA rRNA

the genetic code

mRNA is translated into a sequence of amino acids using a three base pair code. Three bases make up a codon

Most DNA binding proteins interact with the

major grove at a specific sequence of nucleotides.

Which of the following would be the best way to determine the effect of promoter mutations on transcription?

measure specific mRNA expression

mRNA

messenger RNA, encodes proteins transcription of the DNA code that dictates the production of a specific protein. has a distinct structure that is different between prokaryotes and eukaryotes. Common to both types of 5' untranslated section, 3' untranslated section Ribosome Binding site Translation Start site (start codon) Translation Stop site (stop codon)

base modifiers

modify ATCG usually via oxidation

The statement "codons are redundant" refers to the fact that

most amino acids are specified by more than one codon.

direct reversal

mutated base is still recognizable and can be repaired without referring to other strand

second site revertant

mutation is at a different site in the DNA

same site revertant

mutation is at the same site as original mutation

suppressor

mutation that compensates for the effect of the original mutation Suppressor mutations do not necceassrily have to occur in the mutated gene.

primary structure

of a protein is its unique sequence of amino acids Ribosome generates primary structure.

A(n) __________ is composed of DNA.

operator

Which of the following are features of operons?

operator, protein-coding genes, promoter

During translation, amino acids are linked by a peptidyltransferase activity that is due to

part of the 23S rRNA

What functional group do kinases transfer to other molecules as signals of environmental change?

phosphate

why are all genes not expressed at the same time

physical space limitations energy and resource conservation contradictory functions

what are the two major levels of regulation in the cell

post translational regulation regulation of gene expression

Bacteria have evolved ways to prevent phage infection.

prevent phage absorption by modifying pahge receptor or inhibiting receptor binding Prevent DNA injection with specific inner membrane proteins. Restriction-Modification systems Bacteria produces an enzyme (restriction endonuclease) that cuts DNA at specific sequence Bacteria modifies it own DNA so only phage DNA is cut

transduction

process in which bacteriophages carry host DNA from one cell to another. There are two basic types of transduction generalized and specialized

ribosomes

protein machine responsible for protein synthesis Thousands of ribosomes per cell 2 subunits, 30S & 50S facilitate the proper reading of code, the creation of a peptide bond between adjacent amino acids Ribosome is 2/3 RNA, 1/3 protein Ribosomal proteins are generally located on the surface and fill in gaps of the folded RNA; they stabilize the RNA core structure The rRNA in ribosomes function as a ribozyme (RNA with catalytic activity)

homodimeric proteins

proteins composed of two identical polypeptides

antibacterials that target nucleic acid synthesis

quinolones and rifampin/rifampicin

The protein product of the lacI gene is

regulatory protein

Alternative sigma factors are important for controlling expression of

regulons necessary for surviving adverse growth conditions.

replication, transcription, translation

repetitive processes that build polymers of nucleotides or amino acids all have three majors steps, initiation, elongation, and termination

homologous recombination

requires that the two recombining molecules have a considerable stretch of homologous DNA sequences. Bacteria utilize to incorporate foreign DNA, repair damaged DNA, and/or fix "stuck" replication forks Also known as Generalized Recombination

site specific recombination

requires very little sequence homology between the recombining DNA molecules. Lysogenic phages and mobile genetic elements called transposons use site-specific recombination to insert into genomes. Require a specific short sequence recognized by a specific recombination enzyme.

quaternary structure

results when a protein consists of multiple polypeptide chains

spectinomycin (initiation)

reversibly interferes with mRNA interaction with the 30S ribosome.

Guanosine tetraphosphate (ppGpp) serves as a signal to the cell that

ribosome synthesis should be curtailed.

what are the two components of signal transduction

sensor kinase and response regulator

In a two-component signal transduction system, the __________ senses a change in the environment and the __________ changes gene expression.

sensor, response regulator

How is the small ribosomal unit positioned to allow for translation to start at the proper start codon?

shine dalgarno sequence

Accumulation of the heat-shock sigma factor, sigma H, increases at high temperatures because at high temperatures,

sigma H mRNA adopts an unfolded secondary structure.

informational classes of mutations

silent missense nonsense frame shift Note that silent, missense, nonsense and frameshift mutations can occur due to different types of changes in DNA.

deletion

subtraction of one or more bases

genotype

the nucleic acid sequence of the genes an organism carries.

The "coding region" of a gene corresponds to which?

the nucleotides that specify the amino acid sequence of a protein

phenotype

the observable characteristics of an organism that are influenced both by its genotype and by the environment.

recombination

the process by which genetic material is broken and joined with other genetic material. Allows for pieces of DNA from an outside source to be incorporated into a cells genome. Allows for the re-organization of chromosomal material. two main types homologous and site specific recombination

When arabinose is absent

the shape of each AraC subunit is rigid and elongated. Represses gene expression.

Quorum sensing represents a method by which bacteria can measure what?

the size of their populations

conditional mutations

they change the phenotype of organism only under certain environmental conditions.

The prokaryotic ribosome consists of

three RNAs and many proteins

DNA is __________ into mRNA and mRNA is _________ into proteins.

transcribed, translated

tRNA

transfer RNA, shuttles amino acids each amino acid needs at least one . Mammalian cells have 100-110 Bacterial cells have 60 different tRNAs Short 70-90 single stranded ribonucleotide sequences. Sequence, base pairs with itself to create a T-shape, conventionally drawn as a cloverleaf structure.

three mechanisms in bacteria

transformation conjugation transduction

T/F All viral mRNAs are translated by host cell ribosomes!!!

true

A bacterial cell that lacked the Rho protein would be

unable to terminate transcription of some genes.

Transcriptional regulation is normally accomplished by the reversible binding of regulatory proteins. The proteins that activate genes typically bind to sequences ________ of the promoter, whereas those involved in repression usually bind______________of the promoter.

upstream, downstream

(-) ssRNA

viral RNA dependent RNA polymerase

dsRNA

viral RNA dependent RNA polymerase

(+) ssRNA

viral RNA dependent RNA polymerase makes -ssRNA template then +ssRNA genome

to make viral proteins you need

viral mRNA (+ sense, ssRNA). The genome of virus determines the "route" the virus takes to produce mRNA.

RNA retro

viral reverse transcriptase

what are the three classes of bacteriophage

virulent temperate pseudotemperate

naked viruses

viruses that have no membrane

lacZ

β-Galactosidase, converts lactose to glucose and galactose. Can also convert lactose to allolactose (an inducer).

presence of lactose

β-galactosidase (LacZ), cleaves and rearranges lactose to make the inducer allolactose. Allolactose binds to LacI, reducing its affinity to the operator and thus allowing induction of the operon.

lambda DNA replication

λ genome upon entering a host cell is circularized vis "sticky ends" called cos sites DNA is replicated via a rolling circle mechanism producing a long concatemer. The concatemer is cut by a Restriction enzyme, creating the sticky ends. A single unit genome is packaged into the λ capsid.