Exam 3

Coupled transcription and translation

Ribosomes bind mRNA while mRNA is still being created, so that translation begins while transcription is still occurring
- Advantage of lacking a nucleus

Translation initiation

- Initiation factors bind ribosome to the ribosome binding site known as the Shine Delgarno sequence (AGGAGG)
- Begins at start codon
1. 30s subunit binds mRNA
2. IF2 interacts with initiator tRNA
3. fMet-tRNA binds to start codon
4. Association of initiator tRNA with 30s mRNA releases IF3 and allows IF1 to bind
5. 50s subunit binds to 30s complex and GTP hydrolysis occurs

Translation elongation

- A site: charged tRNA entry site
- P site: peptidyl site
- E site: uncharged tRNA exit site
- tRNA binds at A site and peptide bond between amino acid in P and A site

Translation termination

- Uncharged tRNA leaves ribosome
- Stop codon enters the A site and protein release factor enters site
- Binding of release factor releases completed protein from tRNA in P site and causes complex to dissociate

Protein folding

- Primary structure: sequence of amino acids as dictated by gene; ribosome generates primary structure
- Secondary structure: coils and folds in the polypeptide chain; hydrogen bonding of the polypeptide backbone between amine groups
- Tertiary structure: determined by interactions among various side chains (R groups); 3D shape due to R group interactions
- Quaternary structure: results when a protein consists of multiple polypeptide chains

Operon

Unit of genetic material that functions in a coordinator manner by means of an operator, promoter, and one or more structural genes

Promoter

Specific region of a gene that binds RNA polymerase and indicates where to start transcribing RNA

Regulon

System of genes, formed by one or more operons, that have a common regulatory element

Reasons why cells do not express every gene at maximum level functioning

- Physical space limitations
- Energy and resource conservation
- Contradicting functions

Constitutive proteins

Proteins made all the time at a constant rate and are needed at the same level all the time

Post-transcriptional regulation

Control of activity of preexisting enzymes; protein is modified to change its activity

Homodimeric

Protein composed of two identical polypeptides

Negative regulator/repressor

Binds to regulatory sequences in the DNA and prevents transcription of target genes
- Often they block sigma factors of RNA polymerase from binding promoter
- Bind DNA at sequence called operator sequence
- Repressors can require co-factors to repress or be released from binding site

Positive regulator/activator

Binds to regulatory sequences in the DNA and promotes transcription of target genes
- Often acts to recruit or stabilize sigma factor interaction with a promoter
- Binding site is generally upstream of -35, -10 sequence (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

Organization of lac operon

- lacZ: B-Galactosidase; converts lactose to glucose and galactose, can also convert lactose to allolactose (an inducer)
- lacY: Lactose permease; membrane protein that imports lactose from the extracellular environment
- lacI: Lac repressor; protein blocks transcription of the lac operon
- lacO: lac operator; DNA sequence that binds LacI

In the absence of lactose

LacI binds to the operator region and represses the lac operon by preventing open complex formation by RNA polymerase

In the presence of lactose

- B-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

Catabolite repression

- Presence of glucose affects signal inside the cell called cyclic adenosine monophosphate (cAMP)
- Concentrations of cAMP are assessed via cAMP Receptor Proteins (CRP)
- CRP responds to the presence of cAMP

Cyclic adenosine monophosphate (cAMP)

Intracellular second messenger in the signaling cascade synthesized from ATP by adenylate cyclase
- Nutrient sensor

cAMP Receptor Proteins (CRP)

Activator protein/ transcription factor
- Binds next to promoter, stimulates open complex
- Increases transcription of lac and other operons
- Responds to the presence of cAMP
- Acts as activator only when bound to cAMP
- High glucose -\> low cAMP levels -\> CRP inactive

Lac operon expression

Bacterial response to environment

- Bacteria sense the environment; sense levels of nutrients
- Bacteria change gene expression

Two-component signal transduction system

Message relay system composed of a sensor kinase protein and a response regulator protein that regulates gene expression in response to a signal (usually an extracellular signal)

Sensor kinase

- Extracellular receptor for metabolite
- Intracellular communication pathway
- Binds to environmental signal and self-activates via phosphorylation

Response regulator

- DNA-binding protein that regulates transcription
- Activated via phosphorylation by sensor kinase
- Mediates response by altering gene transcription

EnvZ-OmpR System

Two-component system that helps bacteria maintain osmotic homeostasis
- Inner membrane kinase EnvZ self phosphorylates under changes in osmotic pressure
- EnvZ binds OmpR and transfers 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

Sigma factors

Regulate transcription of all genes
- S70 initiates transcription of most genes

Heat-shock response

Coordinated response of cells- changes in the membrane and expression of heat-shock genes- to higher than-normal temperatures
- At 30ºC, secondary structures at the 5 ́-end of rpoH mRNA obscure access to ribosome-binding sites resulting in little RpoH being produced; small amount of RpoH produced is bound by chaperones and degraded
- At 42ºC, heat melts the secondary structure allowing access of the ribosome and translation of the of more rpoH mRNA; heat draws chaperones away from the protein RpoH, allowing them to accumulate and bind RNA polymerase

Anti-sigma factors

Proteins that bind to sigma factors and inhibit their function by blocking their access to the RNA polymerase

Secondary Messangers

Small molecules like cAMP that have regulatory functions
- Can change the activities of regulatory proteins and regulatory RNAs

Guanosine tetraphosphate (ppGpp)

Second messenger that allows cells to handle abrupt changes in nutrient availability

Stringent response

Regulatory mechanism that detects nutrient or environmental stresses and antibiotics
- Allows switching from rapid growth to slower growth
- Nutrient scarcity leaves many ribosomes idle
- Idling ribosomes trigger the synthesis ofppGpp; ppGpp binds RNA polymerase and lowers its ability to transcribe ribosomal RNA (rRNA)genes
- Response causes a decrease in rRNA transcripts made for ribosome assembly, leading to fewer ribosomes and an overall decrease in growth

Quroum sensing

Regulation of gene expression in response to fluctuations in cell-population density
- Quorum-sensing bacteria produce and release chemical signal molecules (autoinducers) that increase in concentration as a function of cell density
- Gram-positive -\> small peptides
- Gram-negative -\> homoserine lactones
- Once a certain number of autoinducers is reached, they bind to receptors that regulate gene transcription

Virus

Non-cellular infectious agent that has genetic material (ss or ds RNA or DNA) inside a protective protein coat and requires a host cell for metabolism and replication
- Lack ribosomes and plasma membrane

Viral properties

- Obligate intracellular parasites
- Completely dependent on host cell for energy acquisition, protein synthesis, and metabolic intermediates
- Acellular, genetic entities
- Can infect prokaryotic cells (bacteriophages) and eukaryotic cells (plant and animal viruses)

Chemical composition of viruses

- Structural Viral Proteins: facilitate transfer of viral genome; protect the viral genetic material; participate in attachment to a susceptible cell; provide structural symmetry; antigenic characteristics
- Viral Enzymes: not structural, but essential for replication; RNA Polymerase; reverse transcriptase

Regression hypothesis

Viruses are small cells that lost their biologic processes

Escape hypothesis

Viruses are retrotransposons that obtained genes encoding capsids

Capsid

Protein coat that encloses a viral genome
- Built from protein subunits (capsomeres)
- Can have various structures

Viral envelope

Membrane, derived from membranes of the host cell, that encloses the capsid, which in turn encloses the viral genome

Virus capsid structure

- Icosahedral: 20 triangular sides; each triangle made up of at least 3 identical capsid proteins; allows small protein to cover a large volume
- Filamentous/helical: 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
- Complex: mixture of icosahedral and filamentous symmetry
- Asymmetrical (irregular): capsid proteins arranged without symmetry; often have an irregular shape.; tend to be larger viruses

Virus envelope structure

- Viruses may have lipid envelope
- Allows fusion to host cell, organelle membrane if host cell is not covered by cell wall
- Not encoded by viral genome
- Proteins embedded in envelope may be coded by virus
- Coats viral capsid as virus leaves cell or organelle

Viral attachment proteins

Protein expressed by virus used for attachment by host cell and interacts with host cell receptor

Viral life cycle

Absorption- attachment of the virus to the host cell
Penetration: viral entry into the cell via fusion of envelope to the plasma membrane, endocytosis, or direct injection
Uncoating: removal of capsid from the genome
Viral genome replication and viral protein synthesis
Virion assembly: packaging of the viral nucleic acid into capsid protein; viral particle is assembled in the cytoplasm of the host
Release: mature virions are released from the host cell via lysis (naked virus) or budding (enveloped virus)

Viral protein synthesis

Viral transcript is translated to produce structural components of the virus
- All viral mRNA's are translated by host cell ribosomes

Polyprotein protein synthesis strategy

Entire, large, multiprotein can be translated all at once; whole genome translated

Splicing strategy

Virus can choose which genes need to be translated; genome is spliced as needed

Segmented genome strategy

Genome is carried in multiple segments; virus can choose which genes are translated
- Can lead to under or over expressing of genes

Viral taxonomy

Based on shape, nucleic acid content, sequence analysis, antibodies, where its located, and/or site of replication

Classification of viruses- biological properties

Host range, mode of transmission, pathogenicity, pathology, vector relationships, and tissue tropisms

Syndrome

Set of physical signs and symptoms that occur together

Latent period

Eclipse: period where the virus copies its genome and produces proteins
Maturation: assembly of viral particle (assembling the capsid around the genome, and incorporating any other required protein)

Burst size

Number of virions released
- Viruses that lyse the cell will be released all at once
- Viruses that bud may be released over a longer period of time

Plaque assay

Method used to measure the number of viral particles present in a sample

Bacteriophage

Virus that infects bacteria

Bacteriophage classes

- Virulent: infection results in cell lysis and the production of many progeny phage (ex. T4)
- Temperate: infection can be virulent or can lead to the maintenance of the virus within the cell in a dormant state, called lysogeny (ex. Lambda)
- Pseudotemperate: infection establishes a permanent relationship with the host; virus is produced and secreted continuously without host cell lysis (ex. SP10)

Virulent phage (T4)

- Infects E. coli
- Only has a lytic life cycle
- Linear double-stranded DNA
- Complex capsid symmetry

T4 life cycle

- Absorption and penetration: tail fibers bind to E. coli LPS during absorption; tail pins make contact with E. coli outer membrane; tail sheath contracts, pushing the tail tube through the outer membrane; T4 Lysozyme makes a small hole in the peptidoglycan and the DNA is injected
- Assembly and release: packaging motor is assembled; dsDNA is pumped into capsid head under pressure using ATP; after head is filled with DNA, tail, tail fibers, and other components are added; once enough virions are assembled, the cell will lyse (burst size is about 100 virions)

T4 genes and gene expression

Large, linear genome divided into genes that are transcribed at early, middle and late time points during infection
- Early genes: nucleases (digest host genome), replication proteins, sigma factors (ensure viral genes are expressed), RNA polymerase modifying proteins
- Middle genes: RNA polymerase modifying proteins
- Late genes: capsid proteins, assembly proteins, release proteins

Temperate phage (λ)

- Uses both/can switch between lytic and lysogenic cycles
- dsDNA genome; linear, with complimentary single-stranded ends; circularizes upon cell entry
- Complex capsid symmetry

λ life cycle

- Lambda absorbs to the LamB porin of E coli; after injecting its genome it can take on one of two pathways: lytic pathway or lysogenic pathway
- Attachment and injection: λ attaches at LamB, a porin involved in maltose transport; utilizes the cell's Mannose transporter system to facilitate entry into the cytoplasm
- λ makes a critical decision: lytic or lysogenic
- Assembly and release: capsid proteins self assemble into viral particles; viral; DNA is packaged in "head full" manner; tails are assembled to full heads; makes protein to depolymerize peptidoglycan; bursts host cell to release progeny phage

λ genome and gene expression

- Early: initiates at PL and PR and terminates after N and Cro; N promotes expression of middle genes
- Middle: initiates from PL and PR; leftward transcription proceeds through the int gene and produces Int (integrase), Xis (excisionase), and regulatory protein CIII; rightward transcription proceeds through the Q gene and produces DNA replication proteins (O, P), and regulatory protein CII; CII vs CIII determine lytic vs lysogenic decision
- Late gene expression: initiates from PR'/Plate; late genes encode phage structural components and lysis enzymes

λ lytic vs lysogenic decision

Lytic vs Lysogenic Switch made up of 3 proteins: CI (represses λ phage gene expression), CII, and CIII (assess cell nutrition)
- CII is a positive regulator and promotes the expression of CI (repressor)
- CIII is an inhibitor of host cell protease that cleaves CII, but is only effective against low levels of protease
- Poor nutrition \= lysogeny; good nutrition \= lytic
- Poor growth media \= high cAMP \= low level of 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
- Rich growth media \= low cAMP \= higher level of protease \= less CII; CI repressor is not made and lytic cycle favored

Establishing lysogeny

- CI represses transcription of all λ genes, except CI
- λ Integrase (Int) and Integration Host Factor (IHF) recognize the attP sequence on phage DNA and attB sequence on the bacterial chromosome and integrate the λ genome into the bacterial chromosome
- After integration, λ is termed a prophage/lysogen

Prophage induction

- CI repressor must be inactivated in order for λ prophage to become lytic
- Only 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

λ DNA replication

- λ genome is circularized vis "sticky ends" called cos sites upon entering the host cell
- 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

Restriction

Prokaryotic destruction of foreign DNA through the activity of restriction endonucleases (enzymes that cleave DNA at specific sites)
- Host cell protects its own DNA

Mosaic nature of genomes

Result of heavy horizontal gene transfer, recombinations, and a variety of mutagenic and DNA repair strategies
- Genomes have mutations (deletions, insertions, duplications, inversions, prophages, and homologs)

Genotype

Nucleic acid sequence of genes an organism carries

Phenotype

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

Mutation

Heritable change in genotype that can lead to a change in phenotype
- Most are silent (no effect on organism); mutations in regions between genes, change in 3rd base of a codon, change of one amino acid into a similar one, or change in protein not needed for growth

Conditional mutation

Change in phenotype of an organism only under certain environmental conditions

Induced mutation

Mutation that occurs due to agents in the environment or deliberate manipulation by humans (i.e., radiation or chemicals)

Spontaneous mutation

Mutation that occurs without external intervention
- Generally result of errors made by DNA polymerase during base pairing

Selectable mutation

Mutation that gives the mutant a growth advantage under certain conditions (ex. antibiotic resistance)

Nonselectable mutation

Mutation that has neither an advantage nor disadvantage over parents

Auxotroph

Organism that is unable to synthesize a particular organic compound required for growth

Wild type

Organism with typical genotype found in nature

Mutant

Strain of any cell or virus that differs from parental strain in genotype

Silent mutation

Mutation that changes a single nucleotide, but does not change the amino acid sequence

Missense mutation

Mutation that changes the amino acid sequence to another

Nonsense mutation

Mutation that changes an amino acid sequence to a stop codon, resulting in a shorter and usually nonfunctional protein

Frameshift mutation

Mutation that shifts the open "reading" frame of the gene, by inserting or deleting a nucleotide

Reversion

DNA mutates back to original sequence; return to normal phenotype

Revertant

Organism that returns to wildtype phenotype

Same site revertant

Mutation is at the same site as original mutation

Second site revertant

Mutation is at a different site in the DNA

Suppressor

Mutation that compensates for the effect of the original mutation

Mutation rates

- Mutations tend to occur at a frequency of 10^-9 per base pair replication event
- Proof reading ability of DNA polymerase lowers error rate to 10^-6 to 10^-7
- DNA repair systems (come in after DNA polymerase) reduce rate of mutation to 10^-9
- Mutation in RNA genomes is typically larger than DNA genomes (due to no RNA repair mechanisms)

Mutagen

Chemical or physical agent that interacts with DNA and causes a mutation

Ames test

Procedure using bacteria to identify potential carcinogens
- Uses bacterial strain auxotrophic for histidine- bacteria cannot grow unless histidine is supplied due to frameshift mutation in hisG gene
- Bacteria is placed on medium with mutagen
- Mutagen causes reversion- bacteria can grow in absence of histidine
- More colonies \= stronger mutagen

Error proof repair

High fidelity repair mechanisms for non-catastrophic damage

Direct reversal

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

Repair of single strand DNA

Damaged DNA is removed and repaired using opposite strand as template

Error prone repair

Low-fidelity repair mechanisms for large-scale DNA damage that interferes with DNA replication
- Cell may use an Error Prone System called the SOS Response
- Wide-scale DNA damage activates RecA; RecA cleaves the LexA repressor protein
- LexA represses genes; in the absence of LexA, repair proteins are transcribed so DNA can be repaired

Error-prone repair pathways

- Light repair: photolyase excise thymine dimers; DNA polymerase I fills the gap; DNA ligase links the new DNA with the old
- 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
- 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

Recombination

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 cell's genome

Homologous/generalized recombination

Exchange of genetic information between homologous DNA molecules
- Requires that 2 recombining molecules have a considerable stretch of homologous DNA sequences

Site specific recombination

Requires very little sequence homology between the recombining DNA molecules
- Require a specific short sequence recognized by a specific recombination enzyme

Generalized recombination

1. RecBCD binds to the end of donor DNA
2. RecBCD unwinds strand until Chi site, where it nicks the DNA and continues unwinding; then RecA filament forms
3. RecA finds homology and mediates strand invasion
4. RuvAB binds at the crossover and carries out branch migration, which extends base paring between donor and recipient strands
5. Endonuclease cleaves one end of displaced recipient DNA loop
6. Displaced ends are ligated to opposite strands