Module 4 Flashcards
where does replication begin on the circular bacterial chromosome
at the origin oriC (an AT rich region that is easy to separate due to few hydrogen bonds)
how is catenation resolved (decatenation)
one of the chromosomes is cleaved by topoisomerase (failure to do this leads to cell death
what is a catenated chromosome
interlocked daughter chromosomes
-replication of circular chromosomes result in the daughter chromosomes interlocking
what is the replisome
a large protein complex that copies DNA
what is a holoenzyme (ex. DNA pol III)
an enzyme and a cofactor
what does the replisome of prokaryotes consist of
helicase, primase and DNA pol III
what protein protects single stranded DNA after it is unwound by helicase
replication protein A (RPA)
what can optimize replication strategies
small genomes, simple genomes (bacteria) - spontaneous mistakes can be beneficial/spread quick
eukaryotes are more complex
compare and contrast replisome components of prokaryotes and eukaryotes
Prokaryotes:
Helicase, primase, DNA polymerase III.
DNA pol III synthesizes both leading and lagging strands.
Okazaki fragments joined by DNA pol I and ligase.
Eukaryotes:
Mcm2-7 helicase (activated by proteins like GINS, Cdc45).
DNA pol α synthesizes RNA primer.
DNA pol ε (leading strand) and DNA pol δ (lagging strand).
PCNA (clamp) and RFC (loading factor) involved.
Shorter Okazaki fragments; RNA primers removed by strand displacement.
Briefly explain how the viral mechanisms of genome replication work in DNA, RNA and retroviruses
DNA viruses replicate using the host’s DNA polymerase machinery for replication and transcription.
RNA viruses need positive strands to be read as mRNA, and the other strands can be used as templates for the other to be read.
Retroviruses convert their RNA genomes into DNA via reverse transcriptase, and integrate the resulting DNA into the host genome.
what is the main difference of DNA polymerase families in bacteria/archaea and eukaryotes
Bacteria and archaea tend to have one specific pol for a very defined function, eukaryotes have several in each family
What are the main groups in the Baltimore Classification?
Double-Stranded DNA (dsDNA)
Single-Stranded DNA (ssDNA)
Double-Stranded RNA (dsRNA)
Positive-Sense Single-Stranded RNA (+ssRNA)
Negative-Sense Single-Stranded RNA (-ssRNA)
Retroviruses (Reverse Transcribing ssRNA)
Reverse Transcribing dsDNA
reverse transcribing, for DNA vs retroviruses
having a partially ds genome that needs to be filled in-> DNA to RNA
retroviruses start with RNA and have to reverse transcribed into DNA
what is an example of a class I virus
T4 phage
how does a T4 phage infect a host
- T4 injects linear DNA into the host cell.
- Host RNA polymerase transcribes early genes (mRNA).
- Host DNA is degraded, and phage DNA is replicated.
- Once phage DNA is replicated, remaining mRNA is transcribed by host RNA pol
- Structural proteins are synthesized and packaged.
- Cell is lysed to release new virions.
how does viral replication of dsDNA differ from ssDNA
dsDNA: The virus genome is double-stranded, so both strands can be used as templates for transcription and replication.
ssDNA: The virus genome is single-stranded, so it must first be converted into double-stranded DNA before replication can occur.
What is the replication strategy of Class VI (Retroviruses)?
Genome type: Positive-sense ssRNA
Reverse transcription: RNA genome is converted to dsDNA by reverse transcriptase.
Integration: The dsDNA is integrated into the host genome by integrase.
mRNA synthesis: Host machinery transcribes proviral DNA into mRNA.
difference in replication between +ssRNA and -ssRNA
Positive: Genome directly acts as mRNA for protein synthesis. Negative-sense RNA is synthesized as a template for new positive-sense RNA genomes.
Negative: Genome is transcribed into complementary positive-sense RNA (mRNA). mRNA is translated, and new negative-sense RNA genomes are synthesized.
how does a double stranded RNA virus such as rotavirus replicate in a cell
- Non enveloped virus enters the host and uncoats, releasing a double-layered particle.
- RdRp transcribes the viral genome inside the particle.
- mRNA translated for protein synthesis, and genome segments are replicated.
- Assembly of viral particles occurs in the viroplasm.
- New virions are released via exocytosis or lysis
what is an example of a +ssRNA virus and a -ssRNA virus
+: SARS-CoV-2
-: Influenza
how does SARS infect a cell (covid)
- Enveloped -> Endocytosis.
- genome is translated
- Proteolysis make protein subunits (machinery)
- The replicase-transcriptase complex (RTC) synthesizes negative-strand RNA and mRNA.
- New genomes are assembled and packaged with structural proteins in the ER
5.Virions are released via exocytosis.
What are the structural proteins of SARS-CoV-2?
Spike (S), Envelope (E), Membrane (M), and Nucleocapsid (N) proteins.
replication strategy of influenza
Attachment: HA binds to host receptors, enveloped virus enters via endocytosis.
Uncoating: Acidification releases negative ssRNA into the cytoplasm.
Nucleus: RNA polymerase makes +RNA and mRNA from the - strand.
Replication & Translation: + RNA replicates the genome; mRNA makes viral proteins.
Packaging & Release: New -ssRNA is packaged into virions, which bud off the host cell.
what is an example of a retrovirus
HIV (enveloped)
How does HIV replication proceed after reverse transcription?
The dsDNA form of HIV integrates into the host genome.
Proviral DNA is transcribed into mRNA to make viral proteins, and ssRNA replicate
New HIV particles are assembled and released via budding
what are the different functions of polymerase
replication
repair
-removng RNA primers or damaged bases
-DNA translesion - inserting base across from damaged nucleotide
positive sense RNA are in a form that can
be recognized by ribosomes to become translated into proteins
are RNA or DNA viruses more subject to mutation/harm
RNA - repair mechanisms are very important
what is a polycistronic mRNA
codes for more than one protein, also known as an operon
open reading frame
An open reading frame has the potential to be translated, it is the portion of the reading frame with no stop codon
what do sigma factors do in bacterial transcription
help RNA pol bind to the promoter and start initiation
what are the stages of translation
initiation
elongation
termination/recycling
what is an advantage to not having a nucleus
transcription and translation can occur at the same time
why is the bacterial ribosome a major antibiotic target
it is 70S meaning it is different then that of eukaryotes, also prevents the translation of mRNA into bacterial proteins
what is a polyribosomal complex
when bacterial mRNA are translated by multiple ribosomes, this complex is formed
which initiation factor in bacterial translation recognizes the start codon
fMet-tRNA
what are the initiation factors that make up the bacterial initiation complex for translation
fMet-tRNA, GTP, initiation factors 1/2/3, 30S subunit
what do release factors do
hydrolyze the polypeptide chain from the tRNA and the disassembly of the ribosome
steps of bacterial initiation
- Initiation factors 1/2/3, GTP, fMet-tRNA and the 30S subunit assemble with the mRNA
- When the 50S subunit joins the complex, IF2 hydrolyzes GTP and IF3 and 1 are released resulting in the 70S initiation complex
- This complex is ready for the next step, elongation
stop codons are recognized by ___
release factors
what are the key elongation factors and what do they do
EF-Tu: delivers tRNA with amino acids to the ribosome
EF-G: helps with translocation (moving mRNA and tRNA through the ribosome
the terminology for regulation of gene expression
Transcriptional regulation: control transcription of gene
Post-transcriptional regulation: control protein production by modifying mRNA. Primarily occurs in eukaryotes
Translational regulation: control translation of mRNA
Post-translational regulation: control activity of protein
after it is produced
what is a regulon
a group of genes regulated by a specific transcription factor in response to environmental signals
the genes have related functions
examples of bacterial transcriptional repressors
Repression by steric hindrance
Prevent interaction of RNA pol with the promoter
Repression by looping
Multiple repressors bind and loop DNA so that RNA pol cannot interact with it
Repression by modulation of an activator
Counteract the activator to reduce attraction of RNA pol
the 3 types of bacterial transcriptional activators
Class I activation
○ Association of an Activator protein upstream of the promoter recruits RNA polymerase to a specific promoter
Class II activation
○ The Activator binds very close to the promoter region where it can interact directly with domain 4 of the sigma factor
Activation by a promoter conformation change
○ Some Activators bind within the promoter region where they act to optimize the alignment of the -35 and -10 elements of the promoter through a conformational change. This facilitates recognition by RNA polymerase.
What is the role of microRNAs in translational regulation?
MicroRNAs (miRNAs):
Regulate translation by binding to mRNA and blocking its translation or promoting its degradation.
-matching game in eukaryotes presented by RISC
In prokaryotes, Hfq proteins help miRNAs regulate sigma S expression by preventing inhibitory RNA structures.
How does post-translational modification (PTM) affect proteins?
PTM can:
Alter protein stability, activity, localization, or signaling.
Phosphorylation is a common PTM that regulates protein function, often in response to signaling pathways.
-kinase transfers phosphate from ATP to protein, phosphatease removes it
what step in translation do antibiotics like to target
elongation
mechanisms of preventing elongation by antibiotics
Preventing delivery of tRNA to A site
Targeting the peptidyl-transferase center (PTC), blocking
peptide bond formation
Blocking translocation
Causing misreading of mRNA (change protein sequence)
how do bacteria resist antibiotics
Prevent antibiotic from reaching ribosome
* Efflux, decreased permeability
Target modification
* Change ribosome structure so antibiotic cannot bind
Target protection
* Produce a protein that removes antibiotic from ribosome
Antibiotic modification/degradation
what criteria does a spontaneous mutation need to have to be selected for in antibiotic resistance
confer resistance to the selecting agent (antibiotic)
not compromise the fitness/normal functioning of the cell
what is the best way to treat HIV
with a drug cocktail that targets it at different points in its life cycle
protease inhibitor
fusion inhibitor
integrase inhibitors
what system do pseudomona infections use to shift between acut and chronic infection
GAC system
how do pseudomonas infect a cell
Detect host signals using membrane protein
Cytoplasmic protein is phosphorylated
Produces microRNAs- control binding of transcriptional regulator to
certain promoters
If microRNA produced, some genes expressed; if no microRNA produced other genes expressed
How does quorum sensing regulate Pseudomonas virulence?
Bacteria communicate via homoserine lactones (HSLs) produced by LasR and RhlR.
HSL binds to transcription factors and makes more HSL (autoinduction)
Hiercharchal: one HSL controls production of another HSL
Each HSL control a virulence factor ( exotoxin A, pyocyanin) when bacteria reach high density.
exotoxin A does what, and pyocyanin does what
inhibits protein synthesis, generates reactive oxygen species
Tat vs Rev in transcriptional control by HIV
Tat binds to viral RNA as it is being synthesized, helping transcription to occur
Rev transports mRNA encoding viral structural proteins out of nucleus, increasing translation
