Exam 2 microbio

Why can the lagging strand only add in sections

The strand has to work 5 to 3 and the direction its unzipping, is the starting point. There for any time anything new unzips it must add in that new space, but in the 5 to 3 direction.

On the lagging strand, DNA polymerase:

• can’t keep going continuously

• so it waits for more DNA to unzip

• then starts a new short piece (an Okazaki fragment)

Each time more of the DNA opens up:

1. A new starting point is made

2. Polymerase builds a short stretch 5′ → 3′

3. It stops

4. Repeats when more DNA is available

DNA polymerase I

• Removes RNA primers on Okazaki fragments (5’→3’ exonuclease activity).

• Fills in the gaps with DNA nucleotides (5’→3’ polymerase activity).

• Has 3’→5’ proofreading exonuclease activity.

• Lower processivity than Pol III → slower, acts fragment by fragment.

DNA polymerase III

• Main enzyme for replicating the leading and lagging strands.

• High processivity → adds thousands of nucleotides without falling off.

• 3’→5’ exonuclease activity → proofreading for accuracy.

• Does not remove RNA primers.

• Lays down bp’s after the primer starting @ 3’ end of the primer

Helicase

• Unwinds the double-stranded DNA at the replication fork

• Breaks hydrogen bonds between complementary bases.

• Creates single-stranded templates for DNA polymerase to copy.

• Works with single-strand binding proteins to keep DNA stable.

Primase

• Synthesizes short RNA primers on the single-stranded DNA template.

• Provides a 3’ hydroxyl group for DNA polymerase to begin DNA synthesis.

• Works on both the leading and lagging strands.

Ligase

• Joins Okazaki fragments on the lagging strand.

• Forms phosphodiester bonds between adjacent DNA fragments.

• Seals gaps in the sugar-phosphate backbone to create a continuous strand.

Promoter

• Starting site during transcription

• a specific DNA sequence where RNA binds and begins to synthesize at the +1 nucleotide (aka the first nucleotide).

Terminator

• A DNA sequence that signals RNA polymerase to stop transcription.

• Causes the RNA transcript to be released from the DNA template.

Replication bubble

• A region of unwound DNA where replication occurs.

• Forms at the origin of replication.

• Contains two replication forks moving in opposite directions.

• Allows simultaneous synthesis of leading and lagging strands

Replication fork

• The Y-shaped region where the double-stranded DNA is separated into single strands.

• DNA polymerase synthesizes new DNA along each template strand.

• Helicase unwinds the DNA at the fork.

• Leading and lagging strands are replicated simultaneously.

Replication

• The process of copying the entire DNA molecule before cell division.

• Produces two identical DNA molecules from one original DNA.

• Involves unwinding the double helix, synthesizing new strands, and proofreading for accuracy.

• Requires enzymes such as helicase, primase, DNA polymerase, and ligase.

Transcription

• The process of making an RNA copy of a DNA template.

• RNA polymerase binds to the promoter and synthesizes RNA in the 5’ to 3’ direction.

• Begins at the start site (+1 nucleotide) and ends at a terminator.

• Produces mRNA, tRNA, or rRNA depending on the gene.

Translation

• The process of synthesizing a protein from an mRNA template.

• Ribosomes read mRNA codons in the 5’ to 3’ direction.

• tRNAs bring the corresponding amino acids to the ribosome.

• Protein synthesis continues until a stop codon is reached.

Stabilizng proteins

• Proteins that bind to single-stranded DNA after it is unwound.

• Prevent the single strands from re-annealing or forming secondary structures.

• Help keep the DNA template accessible for DNA polymerase.

Leading strand

• Synthesized continuously in the 5’ to 3’ direction toward the replication fork.

• Requires only one RNA primer at the origin.

• DNA polymerase adds nucleotides continuously as the fork opens.

Lagging strand

• Synthesized discontinuously in short segments called Okazaki fragments.

• Synthesized away from the replication fork.

• Each fragment requires a new RNA primer.

• DNA ligase joins fragments to form a continuous strand.

Origin of Replication

• The specific DNA sequence where replication begins.

• Helicase unwinds the DNA at this site to form a replication bubble.

• Marks the starting point for leading and lagging strand synthesis.

• Contains sequences recognized by initiator proteins.

Okazaki fragments

• Short DNA fragments synthesized on the lagging strand.

• Made in the 5’ to 3’ direction away from the replication fork.

• Each fragment starts with an RNA primer.

• DNA ligase joins the fragments to form a continuous strand.

Coding Strand

• The DNA strand with the same sequence as the RNA transcript (except T in DNA is U in RNA).

• Also called the sense strand.

• Runs in the 5’ to 3’ direction.

• Not used as the template; RNA polymerase reads the opposite (template) strand

Sigma Factor

• A protein subunit that binds RNA polymerase to help it recognize the promoter.

• Directs RNA polymerase to the correct start site for transcription.

• Dissociates from RNA polymerase after transcription begins.

• Essential for initiating transcription efficiently.

Large ribosomal subunit

• The part of the ribosome that joins amino acids to form a polypeptide chain.

• Contains the peptidyl transferase center, which forms peptide bonds.

• Provides binding sites for tRNA during translation.

• Combines with the small subunit to form a functional ribosome.

Small ribosomal subunit

• The part of the ribosome that binds to the mRNA.

• Reads the mRNA codons during translation.

• Ensures correct base-pairing between mRNA codons and tRNA anticodons.

• Combines with the large subunit to form a functional ribosome.

Why can transcription and translation be coupled in prokaryotes but not in eukaryotes?

• In prokaryotes, there is no nucleus, so mRNA is immediately available to ribosomes as it is being transcribed.

• Translation can begin before transcription is complete.

Operon

• A set of genes all transcribed as a unit and regulated together

• A group of genes under the control of a single promoter and regulatory sequences.

• Includes structural genes, an operator, and a promoter.

• Functions as a unit to coordinate the expression of genes with related functions.

• When the operon is “on,” the genes are transcribed together into a single mRNA.

Regulatory gene

• A gene that codes for a protein (such as a repressor or activator) that controls the expression of an operon.

• Can bind to the operator or promoter to turn the operon on or off.

• Helps the cell respond to environmental changes by regulating gene expression.

Inactive repressor

• A repressor protein that cannot bind to the operator.

• Allows RNA polymerase to bind to the promoter and transcribe the operon.

• Typically occurs when a corepressor is absent (in repressible operons) or an inducer is present (in inducible operons).

• Leads to the operon being turned on.

Activated Repressor

• A repressor protein that can bind to the operator.

• Blocks RNA polymerase from transcribing the operon.

• Usually occurs when a corepressor is present in repressible operons or an inducer is absent in inducible operons.

• Results in the operon being turned off.

Repressor mRNA

• The messenger RNA transcribed from a regulatory gene that codes for a repressor protein.

• Translated into the repressor protein, which can then bind to the operator.

• Controls the expression of the operon by turning it on or off.

• Functions independently of the structural genes in the operon.

Inducer

• A molecule that binds to a repressor and inactivates it.

• Prevents the repressor from binding to the operator.

• Allows RNA polymerase to transcribe the operon.

• Common in inducible operons, such as the lac operon.

Horizontal Gene Transfer

• The movement of genetic material between organisms without reproduction.

• Allows bacteria to acquire new genes from other bacteria.

• Can involve chromosomal DNA or plasmids.

• Main types include transformation, conjugation, and transduction.

Cojugation

• A type of horizontal gene transfer in bacteria.

• Involves direct cell-to-cell contact, usually via a pilus.

• Mostly transfers plasmids, such as fertility (F) plasmids or resistance (R) plasmids. Can sometimes transfer chromosomal DNA.

• Donor survives and keeps a copy of the DNA; recipient acquires new DNA/plasmid and may gain new traits.

Transformation

• A type of horizontal gene transfer in bacteria.

• Bacteria take up free DNA fragments or plasmids from their environment.

• Can result in new traits, such as antibiotic resistance.

• Requires the recipient cell to be competent to take up DNA.

• Donor is usually dead; recipient incorporates the DNA into its genome.

Transduction

• A type of horizontal gene transfer in bacteria.

• DNA is transferred from one bacterium to another by a bacteriophage.

• Can involve chromosomal DNA or plasmids accidentally packaged by the phage.

• Helps bacteria acquire new traits, such as antibiotic resistance.

• Donor may be lysed; recipient receives donor DNA via phage infection.

Plasmids

• Small, circular, double-stranded DNA molecules separate from the bacterial chromosome.

• Can replicate independently of the bacterial genome.

• Often carry genes that provide advantages, such as antibiotic resistance, virulence, or fertility.

• Can be transferred between bacteria through horizontal gene transfer

Virulence

• The degree to which a microbe can cause disease.

• Determined by factors that allow the microbe to infect, damage, or evade the host.

• Includes traits like toxin production, adhesion, and immune system evasion.

• Higher virulence generally leads to more severe infecti

Lysis

• The breaking open of a cell, resulting in its death.

• Can be caused by viral infection, antibiotics, or enzymes.

• In bacteriophages, lysis releases new viral particles from the host cell.

• Often occurs when the cell membrane or cell wall is damaged.

Lysogeny

• A viral life cycle in which the viral genome integrates into the host DNA.

• The virus remains dormant as a prophage and does not immediately kill the host.

• The host cell replicates normally, copying the viral DNA along with its own.

• Can switch to the lytic cycle under certain conditions, producing new viruses.

Capsid

• The protein coat that surrounds and protects the viral genome.

• Determines the shape of the virus (helical, icosahedral, or complex).

• Composed of repeating protein subunits called capsomeres.

• Plays a role in attaching the virus to host cells.

Envelope

• A lipid membrane that surrounds some viruses, derived from the host cell membrane.

• Contains viral proteins that help the virus attach to and enter host cells.

• Found in many animal viruses, but not all viruses have an envelope.

• Makes the virus more sensitive to heat, detergents, and disinfectants.

Endocytosis

• A process by which a virus enters a host cell by being engulfed in a vesicle.

• The host cell membrane wraps around the virus, forming an endocytic vesicle.

• Common entry mechanism for enveloped and some non-enveloped viruses.

• Once inside, the virus escapes the vesicle to release its genome for replication.

Membrane fusion

• A process by which an enveloped virus merges its envelope with the host cell membrane.

• Releases the viral genome directly into the host cytoplasm.

• Does not require vesicle formation like endocytosis.

• Common in enveloped viruses such as influenza and HIV.

Budding

• A process by which enveloped viruses exit the host cell.

• The virus acquires its envelope from the host cell membrane as it leaves.

• Usually does not immediately kill the host cell.

• Common in many animal viruses, such as influenza and HIV.

Exocysosis

• A process by which viruses exit the host cell inside vesicles.

• The vesicles fuse with the host cell membrane to release the virus.

• Usually does not destroy the host cell immediately.

• Common in some enveloped viruses.

Spike proteins

• Proteins on the surface of enveloped viruses that allow attachment to host cells.

• Bind to specific receptors on the host cell membrane.

• Facilitate viral entry by endocytosis or membrane fusion.

• Important targets for the immune system and vaccines.

dsDNA

• Viral genome composed of double-stranded DNA.

• Can be transcribed directly into mRNA by host or viral enzymes.

• Often replicates in the host cell nucleus using host DNA polymerase.

• Includes many bacteriophages and animal viruses like herpesviruses.

ssDNA

• Viral genome composed of single-stranded DNA.

• Must be converted into double-stranded DNA by host enzymes before transcription.

• Can then be transcribed into mRNA for protein synthesis.

• Includes viruses like parvoviruses.

+ssRNA

• Viral genome is single-stranded RNA of positive polarity.

• Can act directly as mRNA and be translated by host ribosomes.

• Does not need to be copied into complementary RNA before translation.

• Includes viruses like poliovirus and flaviviruses

+ssRNA (retroviridae)

• Viral genome is single-stranded RNA of positive polarity.

• Uses reverse transcriptase to convert RNA into DNA after entering the host cell.

• The DNA is integrated into the host genome as a provirus.

• Includes viruses like HIV.

-ssRNA

• Viral genome is single-stranded RNA of negative polarity.

• Cannot be directly translated into protein; must first be transcribed into complementary +ssRNA.

• Uses own RNA-dependent RNA polymerase to produce mRNA.

• Includes viruses like influenza and rabies virus.

dsRNA

• Viral genome is double-stranded RNA.

• Must be transcribed by viral RNA-dependent RNA polymerase to produce mRNA.

• The mRNA is then translated into viral proteins by the host ribosomes.

• Includes viruses like rotavirus.

You are a biochemist at a pharmaceutical company who has been tasked with developing new antiviral drugs. Which stage(s) of the viral replication cycle could you target to prevent a virus from spreading?

• Attachment and entry: block the virus from binding or entering host cells.

• Uncoating: prevent the viral genome from being released into the host cell.

Oseltamivir (a.k.a. Tamiflu) is an antiviral drug that inhibits neuraminidase, which is required for the influenza virus to bud from host cells. Which stage of the viral life cycle does this drug act on?

Release

Amantadine (Symmetrel) is an antiviral drug that prevents uncoating of the influenza virus. Which stage of the viral life cycle does this drug act on?

Entry

Maraviroc is an antiviral drug that prevents HIV from binding to CCR5 receptors on host cells. Which stage of the viral life cycle does this drug act on?

Attachment

Remdesivir is an investigational drug that inhibits RNA-dependent RNA polymerase, a viral enzyme used by +ssRNA viruses to replicate their RNA genome. It received emergency use authorization in May 2020 as an antiviral treatment for severe COVID-19 cases

Synthesis

The influenza vaccine, more commonly called the ‘flu shot’, stimulates a patient’s immune system to produce antibodies that target the virus and prevent infection.

Attachment

Why are enveloped viruses easier to destroy than naked ones?

• Enveloped viruses have a lipid membrane that is sensitive to heat, detergents, and disinfectants.

• Disruption of the envelope prevents the virus from attaching to or entering host cells.

• Naked viruses lack this envelope, so they are more resistant to environmental stresses and disinfectants.

How do vaccines prevent a viral disease?

• Vaccines expose the immune system to a harmless form of the virus (inactivated, attenuated, or part of the virus).

• Stimulates the production of antibodies and memory immune cells.

• Allows the body to recognize and respond quickly if exposed to the real virus.

• Prevents infection or reduces the severity of disease

Latent Virus

• A virus that remains dormant within a host cell without producing new viral particles.

• The viral genome can integrate into the host DNA or persist episomally.

• Can reactivate later, entering the lytic cycle and producing new viruses.

• Examples include herpesviruses.

Prions

• Infectious proteins that cause disease without containing nucleic acids.

• Misfolded prions induce normal proteins to misfold, leading to accumulation in tissues.

• Resistant to heat, chemicals, and standard sterilization methods.

• Cause neurodegenerative diseases such as Creutzfeldt-Jakob disease and mad cow disease.

Prion diseases

Spongiform encephalopathies – BSE, CJD, kuru, scrapies

Types of plasmids

• Fertility (F) plasmids: carry genes for conjugation and formation of pili.

• Resistance (R) plasmids: carry genes for antibiotic or chemical resistance.

• Virulence plasmids: carry genes that increase pathogenicity (toxins, adhesion factors).

• Bacteriocin plasmids: carry genes for proteins that kill other bacteria.

Semiconservative replication

DNA replication produces two DNA molecules, each containing: One original (parental) strand One newly synthesized strand This “half-old, half-new” mechanism ensures genetic information is preserved. ensures each new DNA molecule contains one parental strand that guides accurate base pairing.

Anti-parallel

DNA strands run in opposite directions: One strand runs 5′ → 3′ The complementary strand runs 3′ → 5′ This orientation is critical because DNA polymerase can only synthesize new DNA in the 5′ → 3′ direction. contribute to replication by the anti-parallel strands meaning replication occurs differently on each template: Leading strand: synthesized continuously toward the replication fork. Lagging strand: synthesized discontinuously in Okazaki fragments away from the fork.

Start site for replication

Origin of replication

End site for replication

Termination site

Start site for transcription

Promoter

End site for Translation

Terminator

Start site for translation

AUG start codon

End site for translation

UAA, UAG, UGA stop codons

Silent mutation

Base change does not change amino acid

No effect on protein and phenotype usually unchanged

Missense

Base change results in different amino acid

Protein function may be altered can vary in degree

Nonsense

Base change creates a stop codon

Truncated protein; usually nonfunctional

Frameshift mutations

Insertion or deletion

Shifts reading frame changing all codons downstream

Effect: usually nonfunctional protein, often severe phenotype

Mutagen

impact DNA by adding, deleting, or frameshifting due to an outside source can be chemical or light based

Chemical mutagen

Nitrous acid, bisulfite - removes an amino group from some bases

Ethidium bromide - Inserts between the paired bases

Acridine dyes - cause frameshifts due to insertion between base pairs

Nitrogen base analogs - compete with natural bases for sites on replicating DNA

Radiation mutagen

Ionizing (gamma and X-rays) - form free radicals that causes single or double breaks in DNA

Ultraviolet - cause cross-links between adjacent pyrimidines

Replication in prok’s vs euk’s

Prok’s - single origin, circular chromosome, faster, uses less DNA poly’s

Euk’s - multiple origins, linear chromosome, multiple polymerases’s

Transcription in prok’s vs euk’s

Prok’s - single RNA polymerase with sigma factors to recognize promoters, produce mRNA that requires no processing, and transcription occurs in the cytoplasm, often coupled with translation

Euk’s - have three RNA polymerases (Pol I, II, III) that require transcription factors for promoter recognition, modify mRNA with a 5′ cap, poly-A tail, and splicing to remove introns, and transcription occurs in the nucleus separate from translation.

Translation in prok’s vs euk’s

Prok’s -  use 70S ribosomes (50S + 30S), start translation with formyl-Met, occur in the cytoplasm, and can begin translation while transcription is still ongoing

Euk’s - use 80S ribosomes (60S + 40S), start with Met guided by the 5′ cap, occur in the cytoplasm or rough ER for secreted proteins, and translation happens only after mRNA is fully processed and exported from the nucleus.

Inducible operons

• normally off and are turned on in response to a specific inducer (e.g., lac operon when lactose is present)

• respond to substrate availability

Repressible operons

• normally on and are turned off when a corepressor is present (e.g., trp operon when tryptophan is abundant).

• respond to end-product abundance.

What does it mean for an operon to be “ON'“

The operon is actively being transcribed, so RNA polymerase is producing mRNA and the genes are expressed.

What does it mean for an operon to be “OFF“

Transcription is blocked by a repressor or regulatory mechanism, so the genes are not expressed.

Inducers

bind repressors to turn operon on

Corepressors

bind repressors to turn operon off

Effectors

other molecules that influence activators or repressors

What pathway is a inducible operon CATA or ANA ?

• catabolic pathways, which break down substances for energy or nutrients. Example: the lac operon turns on to metabolize lactose.

What pathway is a repressible operon CATA or ANA ?

• control anabolic pathways, which synthesize essential molecules. Example: the trp operon turns off when tryptophan is abundant.

Structure of viruses

Acellular, Contain either DNA or RNA (not both), which may be single- or double-stranded, Capsid: Protein coat that surrounds and protects the genome; made of capsomeres. Nucleocapsid: The combination of the genome + capsid. Envelope (in some viruses): Lipid membrane derived from host cell, often with glycoprotein spikes used for attachment. No envelope = naked. Helical (rod-shaped) Icosahedral (spherical) Complex (e.g., bacteriophages)

Virus classifications / categories

Genome type - DNA vs RNA, SS vs DS, + sense vs - sense

Capsid structure - Helical, Icosahedral, Complex

Envelope presense - yes = lipid membrane + spikes, none = naked

Host range - Bacteria, Animals, Plants

Mode of replication - How and does it use reverse transcriptase

Disease - classify and located

why are viruses considered non-living

Viruses are not considered living because they lack cellular structure and cannot carry out life processes on their own. They do not have metabolism, cannot produce energy, and cannot replicate independently—they must use a host cell’s machinery to reproduce.

What are the steps to viral replication

Attachment → Entry → Uncoating → Synthesis → Assembly → Release

1. Attachment (adsorption) Virus binds to specific receptors on the host cell using surface proteins or spikes.

2. Entry (penetration) Virus or its genome enters the host cell. Enveloped viruses: fusion or endocytosis Non-enveloped: endocytosis or injection

3. Uncoating Viral capsid is removed, releasing the genome inside the host cell.

4. Synthesis (biosynthesis) Host machinery is used to replicate viral genome and produce viral proteins. Mechanism depends on genome type (DNA, RNA, retrovirus).

5. Assembly (maturation) New viral genomes and capsid proteins are assembled into complete virions.

6. Release New viruses exit the cell: Lysis: cell bursts (common in non-enveloped viruses)

Budding: viruses exit with an envelope (common in enveloped viruses)

How do enveloped viruses enter and leave the cell

typically enter by membrane fusion or endocytosis and are released by budding, acquiring their envelope from the host cell membrane without immediately killing the cell.

How do naked viruses enter and leave the cell

usually enter by endocytosis or direct injection and are released by lysis, which destroys the host cell.

Outline the lytic cycle

Virus immediately replicates, produces new virions, and lyses (kills) the host cell to release them. Outcome: rapid cell death and acute infection.

Considerations when choosing control agent

Type of microbe

Number of microbes

Enviroment

Type of material

Level of control

Safety and toxicity

Time and cost

2 antibiotic classes that inhibit cell wall sythesis

Bacitracin and Beta-Lactams

2 antibiotic classes that disrupt the cell membrane

Polymixin and Polyenes

2 antibiotic classes that inhibit protein synthesis

Macrolides and Mupirocin