BIO 305 Exam 3 Chp 6-9 Flashcards
What are the general structural components of a virus?
Genome, capsid, possible spike proteins or extracellular proteins, possible envelope, and tegument (space between capsid and envelope) if an envelope is present.
What are viroids?
Viroids are the smallest infectious pathogens known. They are composed solely of a short strand of circular, single-stranded RNA that has no protein coating.
What are spike proteins?
Proteins made of glycoprotein that stick up from the virus. Can be used for identification. Can hold capsid and envelope together. Can be used for attachment.
What does icosahedral mean?
20 sided capsid.
What is a filamentous virus shape?
Helical. Longer than wide. Genome or capsid can be helical.
What are asymmetric viruses?
Asymmetrical viruses can have asymmetrical genomes with multiple “chromosomes” or an asymmetrical shape (ex: oval).
What do all viruses need to replicate?
A host.
What is the RNA world hypothesis?
That RNA did everything: storage, structural, and functional.
What is a virus?
Virus=noncellular dynamic particle that infects a host cell to reproduce.
What is a host range?
Host range=number of hosts that a virus can infect.
Can be broad or narrow.
What does promiscuous mean in relation to viruses?
Promiscuous=virus hopping around different hosts.
What is prophage?
Viral DNA integrated into bacteria.
What is a provirus?
Viral DNA integrated into a eukaryote.
What is an endogenous virus?
Endogenous viruses=viruses that get to the germ cells and are passed on to offspring.
What are some positive things that viruses do?
Contribute to nutrient cycling, get rid of microbial blooms, and mediate host population control.
What is a plaque?
Plaque=clear spot in a lawn of cells.
Due to lysing of the bacteria due to a phage.
What is the purpose of viruses having an envelope?
The envelope is usually from the host. Having the envelope means the cell can have an easier time getting into the cell.
How can integrated viral genomes be beneficial?
They can provide resistance to toxins and environmental factors.
For humans, some endogenous virus translates to essential proteins in the placenta.
What is the intracellular replication complex?
Inside the host cell, the virus recruits the cell’s proteins in the replication process to replicate the viral genome.
Where are giant viruses thought to originate from?
Thought to originate from cells, maybe obligate intracellular cells, that underwent reductive evolution.
What are tailed viruses?
The genome is connected to a helical neck that delivers the genome to the host cell.
Six jointed legs stabilize the virus on the host cell.
What are prions?
Prions=aberrant infectious proteins.
Unaffected by treatments that target RNA or DNA.
Includes nucleases and UV radiation.
Prions bind to normal proteins and then change their shape to the prion’s shape.
Describe dsDNA viruses.
Uses its own or hosts’ DNA polymerase for replication.
RNA polymerase can also come from the virus or the host cell.
Group 1.
Describe ssDNA viruses.
Uses hosts’ DNA polymerase to replicate the strand.
Double-stranded DNA can then be read by the hosts’ RNA polymerase.
Group 2.
Describe dsRNA viruses.
Require RNA-dependent RNA polymerase to make mRNA.
A virus may package an RNA polymerase before it leaves the host cell.
Ex: Reoviruses.
Includes rotavirus which causes diarrhea in children.
Group 3.
Describe + ssRNA viruses.
Single-stranded.
Contains + sense strand, the coding strand.
Still needs RNA polymerase to be replicated.
Cannot make more + strand without the - strand.
Can serve as mRNA to make proteins.
Includes coronaviruses.
Group 4.
Describe - ssRNA viruses.
Single stranded.
Contains the - strand which needs to be replicated before translation can occur.
Need to package viral RNA-dependent RNA polymerase.
Often segmented.
Ex: Influenza.
Group 5.
Describe retroviruses.
+ ssRNA.
Has reverse transcriptase which transcribes RNA into double-stranded DNA.
Integrated into the host genome.
Group 6.
Explain pararetroviruses.
Has double-stranded DNA.
Transcribes to RNA.
Then, uses reverse-transcriptase to turn RNA back to progeny DNA.
Reverse transcriptase is packaged into the virion.
Group 7.
Explain the lytic replication cycle.
The virus recognizes the host and attaches to it.
Genome must enter the host cell.
Viral phage progenies must be constructed.
Phage progenies must exit the cell to infect new hosts.
They lyse and break open the host cell as they do this.
Explain the pros and cons of the lytic replication cycle.
Pros: Fast, and can make a lot of virions.
Cons: Kills host cell.
Explain the lysogeny cycle.
The viral genome integrates with the host genome.
Usually site-specific.
The viral genome can cut itself out of the genome and turn back into the lytic cycle if environmental conditions or host health deteriorates.
Explain the pros and cons of the lysogeny cycle.
Pros: Can stay in the host genome a long time undetected so it can spread to daughter cells.
Cons: Cannot leave and go to new hosts. Not actively making virions while in the genome. Not getting genetic mutations as quickly.
Explain the slow release cycle.
Able to enter and exit the cell without killing it.
Single-stranded DNA is made into double-stranded DNA.
Double-stranded DNA slowly forms single-stranded viral progeny.
Uses negative supercoiling.
The virus, with the capsid, leaves the host cell alive.
The host cell is alive but grows and replicates more slowly than uninfected cells.
This is because the virus production took a lot of the host cell’s nutrients.
What type of phages does the lysogeny cycle?
Temperate phages.
What is specialized transduction?
Specialized transduction=transfer of certain genes from the host genome in a newly packaged virus to a new host.
What are the pros and cons of the slow release cycle?
Pro: Does not kill the host.
Cons: Slower and does not produce as many virions (smaller burst size).
List the great variety for viral genomes.
RNA or DNA.
For RNA, can be + or - based on whether it is the sense or antisense strand.
Can be single or double-stranded.
Can be linear or circular.
Whole or segmented.
Segmented=separated into separated “chromosomes”.
Why can’t viruses be related by gene-similarity of host type?
There is such a great variety in genomes that viruses with different genomes may be more closely related. Viruses of different hosts may be more similar than viruses with the same host.
So, to categorize viruses, they are categorized by the type of genome they have and relatedness is determined by the proteome.
What are orthologs?
Orthologs=different species, same function.
What are coliphages?
Coliophages=gut bacteripphages.
How can surface proteins change in relation to viruses?
The cell surface receptors that viruses bind to are very specific. A host can gain immunity by changing its cell-surface proteins.
What is a ghost?
An empty capsid one viral DNA has gone inside the cytoplasm.
What is burst size?
Burst size=size of virus progeny that leave a lysed host cell.
What is dsDNA?
Double-stranded DNA.
What is the difference between early genes and late genes?
Early genes are viral genes that are expressed soonest after translation. Closest to the beginning of translation point. Usually translates for polymerases and transcriptase to continue replication and translation.
Late genes are expressed at the end. They usually code for proteins that will lyse the host cell.
What is transduction?
When a virus takes some of the host’s cell DNA with it.
Explain how the CRISPR system acts as an immune system for bacteria.
When the cell can destroy phage DNA, some of it goes into the CRISPR system to be remembered.
CRISPR DNA becomes crRNA.
crRNA binds to the Cas protein complex.
This complex looks for analogous RNA that binds to genetic material from viruses.
What is the Arc system that viruses use to counteract CRISPR?
Phages have evolved anti-CRISPR proteins called Arc.
Can bind to the Cas system which prevents it to bind to viral genetic material. Therefore, the CRISPR system does not go off.
How do restriction endonucleases protect bacteria from viruses?
Bacteria put methyl groups on some of their bases.
Restriction endonucleases cut DNA without these methyl groups.
Viral DNA genomes do not have these methyl groups so they go off.
How do some viral genomes escape detection from restriction endonucleases?
Viruses with changed genomes such as RNA to DNA escape the restriction endonucleases because in the reverse transcription process methyl groups are added.
What are the 3 main ways that bacteria have resistance to viruses?
Genetic resistance (ex: mutations) Restriction endonucleases. CRISPR.
What is a virome?
Virome=community of viruses in a host.
What is transcytosis?
Transcytosis=human tissue taking up bacteriophages.
What is dysbiosis?
Dysbiosis=deterioration of health due to loss of health-promoting bacteria.
What are some hypotheses for why transcytosis occurs?
Because viruses are inert when not in a host, human cells may move bacteriophages around to help them find hosts.
Having phages inside human cells can provide defenses against bacteria that enter the human cell.
How can phages in the gut be useful?
Bring equilibrium in bacteria numbers to what the human gut can tolerate.
Can modulate the immune system.
Attack biofilms.
Can transfer use genetic material to hosts.
What is tropism?
Tropism=ability for viruses to affect different tissues in eukaryotic multicellular organisms.
Viruses can have a broad range (like Ebola) or a narrow range (like polio).
What is uncoating for viruses?
Uncoating=process by which the capsid comes apart.
Can happen in the cytoplasm or at a specific organelle.
What are ways that viruses can enter animal cells?
The viral membrane binds to receptor proteins and becomes part of the cell membrane.
The genome enters directly into the cytoplasm.
Viruses can be encoded in an endosome.
Endosomes can be broken up by a lysosome, and the genome enters the cytoplasm.
Or endosomes can bring the virus to a specific organelle, like the nucleus.
In the nucleus, viruses can take advantage of DNA polymerase, DNA binding proteins, and transcription factors.
What is an episome?
Episome=DNA that can live autonomously in the cell (like plasmids) or be integrated into the genome.
How can viruses cause cancer?
Oncogenes.
Can encode for abnormal oncogenes that deregulate growth.
Genome integration.
Viruses can encode for factors that stimulate host cell division.
Cell cycle control.
Express viral proteins that mess with host cell cycle controls.
What is an oncovirus?
Virus that causes cancer.
In animal cells, what are the two places where viral assembly most likely takes place?
At the ER and the nucleus for nuclear virions.
Why is it beneficial for viruses to make host cells cancerous?
This is beneficial because the more offspring the host has, the more cells now carry the virus. More viral progeny can be made.
How can low level chronic infections enhance the immune system?
The immune system has to be active and fight infections to gain a better memory and do better at its job.
How do viruses infect plant cells?
Viruses enter through damaged tissue.
Animals that eat plants cause damage and can transmit viruses that way.
Some enter the seed and can infect the next generation.
Enter through plasmodesmata (cell junctions that connect cells). Usually not the first mode of entry but how uninfected cells get infected.
Why can’t plant viruses just enter through the cell wall?
Too thick to penetrate.
What are animal and plant defenses against viruses?
Genetic resistance through mutations.
Harder for animals and crops in monoculture.
Immune system.
Interferons=immune system particles that recognize viral particles such as dsRNA.
The immune system has antibodies to recognize certain viral proteins.
RNA interference:
When translating mRNA, the host RNA complex can recognize the viral RNA and refuse to translate it.
Why must viruses be cultured in a host?
Because viruses cannot be cultured on their own.
What is batch culture?
Batch culture=liquid culture that allows the growth of a large number of viruses.
What is multiplicity of infection?
Multiplicity of infection=virus to host cell ratio.
Can manipulate it to make sure every host cell is infected.
In viral growth curves, what is the eclipse phase?
Eclipse phase=time when no virus progenies are made.
The viral genome is still being delivered.
In viral growth curves, what is the latent phase?
Latent phase=viral progeny have been made but have not yet burst out of the cell.
Easier to see in animal cells because virus progeny usually buds out of the cell.
What is the difference between latent phase and latent infection?
Latent phase=viral progeny have been made but have not yet burst out of the cell.
Latent infection=when a virus maintains its genome in the cell but does not produce any virions.
In viral growth curves, what is the rise phase?
Rise phase=increase in viral progeny from host cells.
What is a lysate?
Lysate=viral particles in suspension.
What is virulence?
Virulence=ability to cause disease.
Why is burst size bigger for animal cells than bacterial cells?
Animal cells are larger and have more resources to make more virions.
What are plaque-forming units (PFUs)?
Plaque forming units (PFUs)=by counting the number of plaques, we can estimate how many viruses there are.
How are viruses cultured in bacteria?
Seen by PFUs. Can generate a lysate for the virions in the burst size.
Why does the method for culturing animal viruses different than culturing viruses from bacteria?
Animal cells are cultured with liquid on top. You could not count the number of plaques because released virions would spread throughout the plate via the liquid.
What is a method for culturing animal viruses?
Infect monolayer.
Allow enough time for viruses to latch onto the host.
Remove liquid layer (aspirate it).
Add gelatin layer.
This will still allow host cells to grow but limit the dispersal of viral progeny.
What are focus methods for culturing animal viruses?
Focus methods are used for cells that are infected by a virus that does not kill them.
Can be visualized by fluorescence using an antibody.
Can be visualized by piles of transformed cells that have infected with oncogenic viruses that turn host cells into cancer cells.
What would happen if a virus did not uncoat inside of a host cell?
No replication would occur.
No viral progeny would be made.
The capsid could be digested and then the genetic material could be released.
What are LTR regions?
LTR regions=long-terminal repeats.
Can be on both ends of the viral genome.
What is a virophage?
A virus that infects a bigger virus.
What are cytopathic effects?
Cytopathic effects=changes in the structure of the cell due to infection by a virus.
What diversity do bacterial chromosomes have?
Circular or linear chromosome.
One or two chromosomes.
What is the assumption between the complexity of an organism and its genome?
More complex organism=more complex genome.
Not always true.
How does the genome compact in the cell?
Uses histones, radial loops, and supercoiling.
What is conjugation?
Conjugation=horizontal transfer of genetic material between two bacteria that takes place over a period of time.
What is site-specific recombination?
Inserting new DNA into a specific spot.
Compare the amount of non-coding DNA between prokaryotes and eukaryotes.
Prokaryotes have a lot less noncoding DNA than eukaryotes.
What is an operon?
Operon=genes operating in tandem.
Controlled by a single promoter.
What is a regulon?
Regulon=functional level of collection of genes and operons that may serve a unified process.
Less common than operons.
What is the nucleoid?
Nucleoid=protein bound domain that holds the genome.
What is positive supercoiling?
Positive supercoiling=overwinding.
Overwinding turns DNA in a clockwise direction and increases the number of twists.
What is negative supercoiling?
Negative supercoiling=underwinding.
Underwinding turns DNA in a counterclockwise direction and lowers the number of twists.
Explain the DNA compaction system in prokaryotes.
DNA is bound by histone-like proteins. The DNA is negatively supercoiled by topoisomerase.
What do topoisomerases and gyrases do?
Topoisomerases and gyrases help add or subtract supercoiling.
Change topology of DNA.
They cause a break in the end, pass DNA through the break and then reseal it.
When does positive supercoiling usually occur?
When DNA is open for replication or transcription. As the fork opens, the DNA will positively supercoil. The positive supercoils have to be removed to continue to open the DNA. Thus, a topoisomerase will be ahead of the fork removing the positive supercoils.
What does topoisomerase I do?
Cleaves one strand.
Thought to remove negative supercoiling.
What does topoisomerase II/gyrase do?
Cleaves both strands.
Uses ATP to generate negative supercoils.
Multisubunit protein.
2 GyrA and 2 GyraB.
What does the antibiotic quinoline target?
Quinoline=antibiotic that targets topoisomerase II.
How does DNA replication occur?
Semiconservative fashion and negatively supercoiled.
What is oriC?
Origin of replication.
What are ter sites?
Termination sites.
There are several and which ones are used depends on the speed of the strands and which one gets to the ter sites first.
What are catenanes?
Catenane=chromosomes are stuck together. Topoisomerase IV (activated by an increase in SeqA) breaks catenanes by a double-strand break and finishes off replicated chromosomes.
What inhibits DNA replication initiation and what promotes it?
SeqA protein inhibits DNA replication initiation.
DnaA promotes DNA replication initiation.
What does DNA adenine methylation (Dam) do?
Methylates the new strand.
What does DnaG do?
It is the primase.
What is the purpose of single-stranded binding proteins (SSBs?)
To protect the ssDNA from being degraded.
Tells cell machinery that this the cell genome and not destroy it.
What does DnaB do?
DnaB=helicase.
What does DnaC do?
It is the helicase loader. This takes the helicases and brings them to the origin.
What does DNA Pol III do?
Main DNA polymerase.
What does DNA Pol I do?
Replace RNA primers.
What are termination utilization substances (tus)?
Terminus utilization substance (tus) binds to these sequences and prevents polymerase escape.
What are plasmids?
Plasmids=small circular DNA.
Negatively supercoiled.
Does not have a critical gene but has useful genes.
Explain plasmid replication.
Autonomous of bacterial chromosome replication.
Can be unidirectional (rolling circle).
Explain the different ways that plasmids make sure they are inherited.
High-copy number plasmids replicate themselves a lot to ensure some plasmids make it to each daughter cell.
Low-copy number plasmids use parC, parR, and parM to build bridges and push copies of plasmids to cell poles. parC is the DNA sequence parR binds to and actin-like filament parM binds to parR and builds a bridge.
How are plasmids transmitted to other cells?
Some transfer by conjugation.
Conjugation can even be between species.
Some are transferred by another plasmid that employs conjugation.
Some are transferred via the transformation of DNA in solution.
What is a microbiome?
Microbiome=microorganisms live in complex communities of multiple species.
What is metagenomics used for?
Metagenome is used to identify species, activities, and contributions, of organisms in a microbiome.
What are OTUs?
Operational taxonomic units.
Why are OTUs used?
This is a conservative approach when using metagenomics because you can’t always get down to the species level.
What is a contig?
Contig=overlapping sequences.
Explain shotgun sequencing.
Used in metagenomics.
DNA is fragmented into pieces and pieced back together by finding overlapping segments.
Why can single-cell genomics be useful?
Uses a single cell.
Single-cell genomics can help with low abundance organisms and will help with single taxon assignment.
What’s the difference between plasmids and secondary chromosomes?
Plasmids do not contain any essential genes while secondary chromosomes do.
Plasmid replication is autonomous while secondary chromosome replication is lined up with the primary chromosome’s replication.
What is transformation?
Transformation=horizontal gene transfer between microbes.
What are purines?
Purines=adenine and guanine.
What are pyrimidines?
Cysteine and thymine.
How does RNA differ from DNA?
Uracil replaces the pyrimidine thymine.
Has a hydroxy group at 2’.
RNA can loop on itself to cause hairpins.
RNA is less stable than DNA.
RNA can be involved in structure and function in addition to holding information.
Because of chemical differences, enzymes that work on DNA only work on DNA.
RNA and DNA can hybridize together when complementary.
Needed for gene expression.
What 3 things are needed for DNA replication initiation?
DNA replication cannot be synthesized until:
The origin is methylated.
SeqA dissociates.
DnaA-ATP levels rise and bind to the origin of replication.
Who experimentally figured out the genetic code?
Nirenberg, Matthaei, and Singer experimentally identified the RNA genetic code.
Figured out the codons, how they are read, and which amino acid was synthesized.
What is RNA polymerase made out of?
Core enzyme and sigma factor.
What makes up the core enzyme of RNA polymerase?
Always the same: 2 Alpha subunits, 1 beta subunit, and 1 beta-prime subunit (B’).
What is the purpose of the sigma factor of RNA polymerase?
Sigma factor=binds to the promoter for initiation.
Many different sigma factors.
Binds to beta and B’ subunits.
What are the components of genes?
Enhancer and repressor cells away from the promoter.
In eukaryotes, these can even be on different chromosomes.
Promoter(s).
Transcription start.
Open reading frame (ORF)=area between transcription start and end.
Termination sequence.
What are consensus sequences?
Found in promoters and signals that the following sequence is a promoter.
What does polycistronic mean?
Polycistronic=multiple genes in an mRNA strand.
What does polycistronic mean?
Monocistronic=one gene in an mRNA strand.
Explain transcription initiation.
Sigma factor scans DNA for matching promoter.
Core enzyme binds to promoter and forms closed complex.
RNA polymerase twists DNA open to form the open complex.
Unwinding initiates with rNTPs and the sigma factor is released.
What two different types of termination is there for transcription?
Uses Rho (rho-dependent) and NusA (rho-independent) contact RNA polymerase to stop transcription.
Explain rho-dependent transcription termination.
RNA polymerases code for mRNA that the rho protein recognizes. Then the RNA polymerase codes for a sequence that causes a stem-loop and the RNA polymerase stutters. Rho catches up to the RNA polymerase, pulling itself up the mRNA knocks the RNA polymerase off.
Explain rho-independent transcription termination.
Terminator sequence causes a stem-loop to form.
RNA polymerase stutters.
NusA protein binds and takes RNA polymerase off the sequence.
What does the antibiotic actinomycin D bind to?
Actinomycin D intercalates between G and C bp.
What does the antibiotic rifampicin bind to?
Rifampicin blocks mRNA exit from the core enzyme.
What does small RNA do?
Small RNA (sRNA)=involved in stabilizing or disrupting structures. Like stem loops.
What is tmRNA?
tmRNA=properites of mRNA and tRNA.
What is catalytic RNA?
Ribozymes.
Why does RNA degrade so quickly?
Cell does not want extra RNA around possible coding for proteins it doesn’t need at the moment so they degrade it by RNases.
RNA is also quickly degraded because it is ss and has no protected ends.
Why do tRNA and rRNA not degrade as quickly as mRNA?
Not all ss.
Can fold on itself.
Has modified bases.
What do regulatory proteins for transcription do and what are some examples?
Regulatory proteins control the initiation of bacterial transcription.
Ex: Enhancers or repressors.
What do repressors do?
They bind to DNA and decrease how easily mRNA can access the gene.
What do activators do?
They stimulate transcription.
What do inducers do?
Inducers bind to activators and repressors
When inducers bind to repressors, they can cause them to fall off and increase transcription.
When corepressors bind to repressors, they decrease transcription.
When inducers bind to activators, they can help bind and increase transcription.
Inducers, called inhibitors, can bind to activators and remove them from the gene to decrease transcription.
What does a kinase do?
Kinase=enzyme that phosphorylates something else.
How can signal transduction pathways control transcription?
Phosphorylation of several proteins can lead to one that interacts with the DNA and upregulates or downregulates transcription.
What does B-galactosidase do?
B-galactosidase can break down lactose into galactose and glucose.
What happens in the cell if there is lactose and little B-galactosidase?
Lactose is broken down into allolactose.
What does allolactose do in the lac operon?
It induces the repressor.
Used when there is lactose and little B-galactosidase.
What does cAMP do?
It is the activator for the lac operon when glucose is low.
Describe the lac operon when there is glucose and no lactose.
Lac operon repressed and no activation from cAMP.
Describe the lac operon when there is lactose and glucose.
Lac operon induced (so repressor is removed). No activation from cAMP.
Describe the lac operon when there is lactose and no glucose.
Lac operon induced and activation from cAMP.
What is the 50S subunit made out of?
50S=33 proteins and 2 rRNA (5S and 23S).
What is the Shine-Dalgarno sequence?
Shine-Dalgarno sequence=purine rich sequence in the leader sequence.
Describe the initiation of translation.
30S subunit 16S RNA binds to Shine-Dalgarno sequence.
Initiation factors (IF1 and IF3) help load the 30S ribosome.
IF2-GTP brings in the initiator tRNA.
50S ribosome associates with the 30S subunit after IF1 and IF3 are released.
A, P, and E sites on ribosome.
IF3 binds in the E site.
Describe the elongation of translation.
EF-Tu-GTP brings the next charged tRNA to the A site.
EF-Tu-GTP is hydrolyzed and leaves.
Peptidyltransferase transfers peptide chain to new charged tRNA in the A site.
Translocation occurs with the help of EF-G-GTP.
tRNAs shift over as the ribosome moves to the next codon.
EF-G-GDP leaves and the A site is ready for the next tRNA.
Describe termination of translation.
Release factors RF1 and RF2 bound to the stop codon, stop translation.
RF3 and Ribosome recycling factor help reset the ribosome for the next mRNA.
What does the antibiotic streptomycin do?
Streptomycin (aminoglycoside) binds to 16S rRNA to make translation sloppy.
What does the antibiotic erythromycin do?
Erythromycin binds to the 23S rRNA of the 50S subunit and interferes with peptide bond formation.
What is a polysome?
Polysome=multiple ribosomes on a single mRNA.
Though coupling can happen in prokaryotes why is still rare? Why would prokaryotes want them to be separate processes instead of completing them all in 1 step?
mRNA may have to form a secondary structure first or proteins need to bind to it to regulate its translation.
Why were chaperone proteins originally called heat shock proteins?
Seen increased expression with increasing temperature. Needed at all times but even more so at higher temperatures because some proteins may start to be denatured.
Compare how GroEL and GroES fold proteins vs DnaK.
GroEL and GroES form a barrel shape and the protein fits inside.
Can even be attached together to form a bigger barrel.
DnaK (Hsp70) clamps onto a protein to help with structure.
What is the triage of folding for proteins before they are digested and discarded?
Protein tries to fold itself.
Chaperones, such as DnaJ and K, can bind to attempt to fold a protein correctly.
GroEL helps if the protein is still not folded correctly.
If unable to fold correctly, it binds to the ATPase subunit of bacterial proteasome-like protease.
Recognizes normally protected hydrophobic domains of a misfolded protein.
If hydrophobic regions are exposed, it means the protein has not folded correctly.
This happens if the protein cannot be folded correctly and the protease breaks it down.
What special shape is associated with outer membrane proteins (OMP)?
Beta-barrel shape.
What is a holoenzyme?
Core protein of RNA polymerase and sigma factor.
What is the importance of the tRNA wobble?
Wobble=tRNA being able to recognize different codons. Wobble is due to the curvature of the anticodon loop and special base inosine.
The wobble allows for fewer tRNAs to be needed.
What is attenuation?
Using translation to regulate transcription.
What is acetylation?
Adding acetyl to the protein to increase its overall stability.
What is lipidation?
Adding lipids to proteins.
Can help the protein stick more in the hydrophobic membrane.
What is glycosylation?
Adding mono- or polysaccharides to proteins to make glycoproteins.
What is translocation?
Translocation=ribosome moving along mRNA or protein being moved to different cell compartments.
What do BAM proteins do?
BAM=beta-barrel assembly machine.
Facilitates OMP insertion to the outer membrane.
Describe replication initiation.
DNA replication cannot be synthesized until:
The origin is methylated.
SeqA dissociates.
It binds tightly to hemimethylated strands but not to double methylated strands.
DnaA-ATP levels rise and bind to the origin of replication.
DnaA initiates replication at the origin.
It binds to the 9mer site and that opens up the 13mer site.
DnaB (helicase) is brought the opened replication fork by helicase loader (DnaC).
The loaders drop off the helicase and then leave.
DnaG (primase) comes to the replication fork and makes RNA primers.
DNA Pol III is brought to the replication site and starts elongation.
Describe replication elongation.
DNA Pol III is the main polymerase doing elongation.
DNA Pol I is an RNase that removes RNA primers.
It uses the end of the 3’ of the last primer as its own primer.
DNA ligase repairs nicks Pol I can’t fix.
Topoisomerase /DNA gyrase removes positive supercoils ahead of helicase.
Describe replication termination.
Several ter sites.
Which one is used depends on the speed of the strands.
Tus protein binds to terminator sequences to make sure DNA polymerase stops.
FtsK travels along KOPS (FtsK-orienting polar sequences) to the ter sites where it can separate the chromatids into the poles of the cell.
Describe plasmid replication.
Using rolling circle replication which is unidirectional.
Replication is autonomous.
RepA nicks the + strand at its origin.
It pulls the 5’ + strand away leaving the 3’ end open to act as a primer.
RepA continues to pull away as a new + strand is synthesized.
The old plus-strand needs a primer and DNA ligase to fix the nick in the backbone that is left at the end.
Describe transcription elongation.
NusG controls the rate of elongation.
Topoisomerases remove positive supercoils.
rNTPs are added and energy for attachment comes from dephosphorylation of 2 Ps.
Describe translation initiation.
IF3 binds to the 30S subunit to separate 30S and 50S subunits.
This allows other proteins to add to the subunits.
IF1 binds to the A site, blocking it.
mRNA joins with the 30S and aligns on the 16S RNA sequence.
Finds Shine-Dalgarno sequence and then the first part of the first codon.
IF2 is complexed to GTP.
IF2 + GTP brings fMet-tRNA to the P site.
GTP hydrolyzes and all initiator proteins leave the ribosome.
50S subunit binds to 30S subunit and now they are locked and loaded.
Describe translation elongation.
EF-Tu-GTP brings the next charged tRNA to the A site.
EF-Tu-GTP is hydrolyzed and leaves.
Peptidyltransferase transfers peptide chain to new charged tRNA in the A site.
Charged tRNA moves to P site and vacant tRNA moves to E site.
EF-G-GTP attaches to the now vacant A site.
To move the ribosome to the next codon, GTP is hydrolyzed to cause a ratcheting of the 30S subunit.
Now, the next codon is ready and the next tRNA can join.
Uncharged tRNA in the E site is kicked out due to conformational changes caused by a new charged tRNA in the A site.
Describe translation termination.
Release factor (RF1 or RF2) binds to A site when the stop codon is reached.
Release factors cause uncharged tRNA in the E site to leave.
Also activates peptidyltransferase to cut the peptide bond and release peptide.
Ribosome disassembles after protein is gone.
Release factor 3 gets out RF1 and RF2.
Ribosome recycling factor (RRF) and EF-G-GTP enter the A site.
The 50S and 30S undock from each other after GTP hydrolysis.
IF3 binds to 30S subunit and uncharged tRNA in the P site and mRNA leave.
Describe protein secretion to the inner membrane.
Signal recognition particle (SRP) pauses translation and brings protein to SecYEG for insertion in the membrane.
The protein can be fully translated before secretion or the processes can be coupled together.
Some proteins don’t need SecYEG to be inserted into the membrane.
Describe protein secretion to the periplasm.
Protein is fully translated in the cytoplasm.
Trigger factor is a chaperone protein that grabs the new protein and keeps it in a lightly folded fashion.
Trigger factor brings the protein to SecB.
SecB unfolds the protein and brings it to SecA.
Having an unfolded protein is important because it easier to transfer a far more flexible unfolded protein than a folded one.
SecA is associated with SecYEG.
SecA ATPase plunges about 20 amino through SecYEG.
ATP hydrolysis causes SecA to withdraw.
SecA binds to more ATP and then pushes 20 more amino acids of the protein through.
LepB in the periplasm clips the end of the protein to make it mature.
Why are some proteins folded before being transferred to the periplasm?
To carefully control the addition of cofactors.
Transported by the twin-arginine translocase (TAT) system.
TAT recognizes the twin-arginine motif in the N-terminus of the protein.
Describe protein secretion to the outer membrane.
Outer membrane proteins have a beta-barrel shape.
Use a SecA dependent mechanism for periplasm delivery.
Beta-Barrel Assembly Machine (BAM) facilitates insertion in the outer membrane.
Outer membrane proteins (OMPs) can be protected by periplasmic chaperones proteins as they travel to the outer membrane.
Describe protein secretion to outside of the cell.
Several types of secretion systems.
Type I uses the ABC cassette system to push proteins out of the cell.
There is a protein tunnel so they never touch the periplasm.
Why is the evolution in microorganisms faster than it is in humans?
Their mutation rate is the same but their generation time is so much shorter so their evolution occurs faster.
What are point mutations?
Point mutations=changes one nucleotide.
What is a transition point mutation?
Transition=pyrimidine with a different pyrimidine or a purine with a different purine.
What is a transversion point mutation?
Transversion=purine swapped with a pyrimidine or a pyrimidine swapped with a purine.
What is a missense mutation?
Missense mutation=changes one nucleotide and a different amino acid is coded for.
What is conservative missense mutation?
The new amino acid is similar to the old one.
What is a nonconservative missense mutation?
The new amino acid is not similar to the old one.
What is a nonsense mutation?
Nonsense=changes one nucleotide so that a stop codon is coded for early.
What is a frameshift mutation?
Frameshift mutation=deletion or the addition of 1-2 nucleotides that changes the reading frame.
What is a duplication mutation?
Duplication=DNA sequence is copied and inserted into the genome.
What is an inversion mutation?
A flip of the DNA.
What is a transposition mutation?
Transposition=movement of sequence fragments from one location to another.
What is a reverse mutation?
The mutated sequence goes back to the original sequence.
What is a loss of function mutation?
Loss of function mutation=decrease or eliminate the activity of the protein.
What is a gain of function mutation?
Gain of function mutations=mutations where protein function has expanded or now has a different substrate specificity.
What is a knockout mutation?
Knockout mutation=mutation that causes elimination of protein function.
What are the steps to transfer F-factor?
Transfer of F factor:
Pili is made between the two cells.
Pili contracts and brings them closer together.
Pili contraction activates the physically bound relaxosome complex.
Relaxosome complex binds to oriT.
Made a single-stranded bubble in oriT.
Another relaxase molecule.
This unwinds the DNA and separates the strand that will be transferred and the one that will not be transferred.
The original relaxase nicks the backbone in the nic site of oriT.
A second relaxase takes the 5’ of the nicked strand through the T4SS transfer system (the pilus).
Strands in both cells are replicated using rolling circle replication.
The one in the donor cell starts replication before all the other strand is transferred into the recipient cell so it can be used as a primer for DNA polymerase.
What is F-factor?
F-factor is a plasmid that has machinery and genes to complete conjugation.
What is methyl-mismatch repair?
Error-proof replication system.
Methyl mismatch repair=compares methylated parental strand to daughter strand.
A mismatch causes a bump in the strand that can be recognized.
Involves Mut proteins S, H, L, and UrvD.
MutS binds to the nearest GATC site.
MutH cleaves unmethylated strand 5’ to GATC.
UrvD (a DNA helicase) unwinds the nicked strand.
Exonuclease removes damaged strand.
Pol I synthesizes a replacement.
Ligase repairs nicks in the strand.
What is base excision repair?
Error-proof replication system.
Base-excision repair:
Glycosylases cleave the bond connecting the damaged base to the rest of the nucleotide.
Now there is an apurinic or apyrimidinic site.
Endonuclease cleaves phosphodiester bond.
Pol I fills in the gap.
Ligase seals nick.
What is photoreactivation repair?
Error-proof replication system.
Photoreactivation=repairs DNA from UV exposure by using light in low wavelength in visible color range, for energy to break thymine dimer and cyclobutane ring.
No bases are excised.
What is nucleotide excision repair?
Nucleotide excision=uses UvrABCD to recognize helical destabilization by pyrimidine dimers to remove a patch of bases.
What is homologous recombination repair?
Error-proof replication system.
Homologous recombination:
Double strand break occurs.
Endonucleases digest 5’ of break and leave 3’ overhangs.
RecA brings homologous strands on another chromosome to the double-strand break.
Repair polymerases use the undamaged homologous DNA as a template.
DNA synthesis fixes the broken strands.
DNA ligase fixes DNA nicks.
What is SOS repair system?
Error-prone replication system.
SOS response=DNA repair systems that introduce mutations into the genome.
RecA is activated because the levels of ssDNA surpass the threshold for activation.
RecA has corpotease activity.
Corpotease activity=stimulates autodigestion of the LexA the inhibitor at the promoters for DNA repair enzymes.
When LexA is gone, DinB, DNA polymerase IV that lacks proofreading ability, and UmuDC, DNA polymerase V that lacks proofreading ability are expressed.
Cell division stops during SOS response.
sulA inhibits FtsZ.
Pol IV and Pol V complete translesion base replication and remake DNA.
Once DNA is made, RecA stops corpotease activity and LexA inhibits the expression of SOS DNA proteins again.
What is nonhomologous endjoining replication system?
Fast-growing bacteria do not use this.
Ex: E. coli.
Slow-growing bacteria use this.
Double strand break occurs.
Ku protein binds both ends and recruits LigD.
LigD has a polymerase and exonuclease activity.
Fills in or removes overhangs and ligates strands.
The result usually leads to missing DNA.
What would happen if cells lacked Dam (DNA adenine methylase)?
Error-proof pathways could not tell which strand was the parental strand and which is the daughter strand.
Neither is methylated.
The repair system will randomly pick one strand as the parent strand. 50% chance it will be right.
What about if Dam (DNA adenine methylase) was overexpressed?
Error-proof pathways could not tell which strand was the parental strand and which is the daughter strand.
Both are methylated.
The daughter strand will be methylated a lot faster.
The repair system will randomly pick one strand as the parent strand. 50% chance it will be right.
Methylation can inhibit transcription.
What is the role/function of methylation?
Inhibit restriction enzymes from cutting genomic DNA.
Regulate transcription rate.
Allow repair systems to know which strand is the parental strand.
What is conjugation?
Conjugation=exchange of genetic material via cell-cell contact.
What is high-frequency recombination (Hfr)?
F-factor that integrates into the chromosome. Able to transfer extra genes to the recipient cell if there is a homologous loci.
Why is Hfr rare?
Need homologous region on recipient chromosome.
What is the difference between oriV and oriT on F-factor?
oriV is used for normal replication in the cell.
oriT is used for conjugation.
Why is conjugation rare?
Conjugation doesn’t happen all the time.
May require an environmental cue.
The recipient may lose the plasmid if there is not reason to keep it.
Why do cells go through gene transfer?
Food.
Material to repair DNA.
Acquire novel genes.
What are transformasomes?
Transformasomes=protein complexes to pick up DNA from the environment.
What are the steps for natural transformation?
Growth-phase dependent.
Composed of a binding protein that captures extracellular DNA.
A pilus can be used that retracts by removing the base of the pilus.
Then, there are proteins to build a transmembrane probe like ComEA that ratchet DNA into the cell.
dsDNA unwinds, one strand is degraded and the other is put into the cytoplasm through the ComEC protein.
SSBs protect ssDNA in the cytoplasm.
RecA replaces SSBs and takes ssDNA to the chromosome for integration in homologous regions.
What is a competent cell?
Competent cell=cell with completed transformasome.
More readily available to get new DNA.
How can you get cells to artificially complete transformation?
An artificial way to trigger transformation can be done by shocking the cell or perturbing the membrane by chemicals or electrical methods.
Do cells just take up any DNA they come across?
No, some only take up specific sequences.
How does transduction occur?
It is an accidental process by phages when they take up some host cell DNA.
What is generalized transduction?
Generalized transduction=can take any gene from the host cell and place it in a recipient.
How does generalized transduction occur?
Occurs when host cell DNA is homologous to viral DNA.
More likely to occur is host cell DNA is fragmented. (Like possibly during SOS response).
What are transducing particles?
Transducing particles=cell particles that only contain host DNA.
When transferred to a new host, they cannot make more viruses, but they can recombine with the new host genome.
What is specialized transduction?
Specialized transduction=transfer only genes that are close to prophages.
How does specialized transduction occur?
It occurs due to aberrant excision; when prophage excises themselves from the genome, they may take some host genomes with them.
What can specialized transduction lead to in the recipient cell in its genome?
Partial diploids.
What is the innate immune system in bacteria?
Restriction and methylation systems.
What do restriction endonucleases do?
Restriction endonucleases make staggered nicks the backbone at specific sequences to destroy potentially harmful DNA.
Why are CRISPR and restriction and methylation systems not perfect?
The cell wants to allow some new DNA to come in because it can still have a benefit.
How can phage DNA evade restriction-modification screening system?
Their restriction sites might be methylated before they encounter restriction enzymes.
Flood the cell with the DNA.
Viral DNA can get methylated in the cell.
What is the active immune system in bacteria?
CRISPR.
Describe what is in the CRISPR locus.
CRISPR locus has short repeats separated by spacers, which are the pieces of viral DNA, cas genes, and subtype genes.
What are the 3 stages of CRISPR (just their names not what is involved in each)?
Adaptation
crRNA processing
Interference
What does the adaptation phase in CRISPR involve?
Cas 1-Cas 2 complex cleaves foreign DNA near the protospacer adjacent motif (PAM).
What does the crRNA processing phase in CRISPR involve?
Protospacer is placed into the lead of the CRISPR array.
CRISPR is transcribed to make pre-crRNA.
Pre-crRNA is broken up according to the spacer and makes mature crRNAs.
crRNAs are attached to a Cas protein.
What does the interference processing phase in CRISPR involve?
crRNAs guide Cas proteins to the homologous region of target DNA.
What is RNA interference?
RNA interference=RNA binding to RNA to make dsRNA.
For CRISPR, If new phage DNA is found, how does the cell know what the protospacer is and what piece of DNA to cut?
CRISPR recognizes protospacer adjacent sequence as a general recognition site and cuts the DNA next to it.
Why would bacteriophages containing CRISPR-Cas arrays be beneficial?
To destroy competition in the cell.
One thing to remember is that when a virus infects the cell, it becomes more immune to other viral infections.
The virus can help the cell fend off other viruses.
Recognizes the CRISPR-Cas 9 in bacteria and protects itself from it.
Evolved through transduction.
Does a double-stranded sequence have to be fully methylated to be protected from restriction endonucleases?
No, it only has to be hemi-methylated.
In the CRISPR system, how does the cell know self from foreign DNA in the spacers?
The spacers do not contain the Protospacer adjacent motif (PAM) sequence that foreign DNA has.
What are transposons?
Transposons are mobile genetic elements that carry genes for transposition and other genes.
What sequence must all transposons have?
Transposase, the enzyme that cuts the transposon out and moves it to a new location.
What are the general steps of transposition?
Transposase binds to inverted repeats on the sides of the transposon.
Makes a cut like restriction enzymes.
Transposon moves and new inverted repeats are made.
What is the difference between replicative and nonreplicative transposition?
In replicative transposition, the transposon is copied, and then it moves. So, it is in the old spot and the new spot.
In nonreplicative transposition, the transposon is cut from the old spot and moved to a new spot. The transposon is only in the new spot.
What is conjugative transposition?
Conjugative transposition=transposons move from one cell to the other via conjugation.
What is one major concern with conjugative transposition and the genes it moves?
Can result in moving antibiotic-resistant genes.
Some transposon movement is triggered by antibiotic-caused damage.
Are transposons autonomous or controlled by the host cell?
Autonomous.
What are inverted DNA sequences?
Inverted DNA sequence=DNA sequence identical to a downstream sequence that is separated by nucleotides.
These are added when a transposable element jumps.
Usually on the opposite strand.
What is an ortholog?
Ortholog=different species, same function.
What is a paralog?
Paralog=different species, same function.
Why are transposons of interest in mutation assays?
Only interrupt one gene or operon.
Can be inserted in random places in the genome.
Can have antibiotic-resistant genes which can be used to remove all non-mutants from the pool.
Can truncate a gene if it contains termination sequences.
Why are duplications of interest in evolution?
Can keep the function of the old gene and allow divergence in function in a duplicated gene.
What is genome reduction?
Genome reduction=large scale loss of genes.
What are pseudogenes?
Pseudogenes=genes that no longer encode for a functional protein.
The function can be lost of invading a new environment or using different nutrients.
What are the 3 major ways that horizontal gene transfer can be done?
Conjugation.
Transduction.
Transformation.
What are homologs?
Homologs=genes with shared ancestry and sequence similarity.
