Unit 2 genetic

Lec 9: Bacterial and viral systems

What is a prototrophic bacteria?

A bacteria that can synthesize all essential compounds required for growth using basic minimal medium (sugar + inorganic salts).

What is a auxotrophic bacteria?

A bacteria that lack at least one enzyme necessary for growth (such as amino acid biosynthesis) and must be supplemented in the medium

How does a haploid genome affect the expression of genetic mutations in bacteria?

Because bacteria are haploid (having only one copy of each gene), all mutations are expressed directly. There are no dominant or recessive masking effects from a homologous chromosome.

What is replica plating and what is it used to identify?

A laboratory technique where a bacterial culture is first grown on a fully supplemented master plate, and then its colonies are transferred via a sterile velvet surface to new plates with different media conditions. It is used to identify and isolate auxotrophic mutants based on their inability to grow on specific lacking media.

What is a plasmids?

A small, circular, extrachromosomal dna that can be carried by bacteria and replicate independently of the bacterial genome. They often carry adaptive genes, such as antibiotic resistance genes.

What is an episome?

It is a plasmid that can replicate independently and can also integrate into the bacterial genome

What is the F factor?

The fertility factor is an E. coli episome that contains the genetic instructions necessary for regulating cell plasmid transfer/mating (conjugation) between cells. It encodes for replication genes, insertion sequences (for genomic integration), an origin of replication (ori), and an origin of transfer (oriT).

What are the three primary mechanisms of horizontal gene transfer in bacteria?

• Conjugation: Direct transfer of DNA between two cells via a temporary physical connection.

• Transformation: Direct uptake of naked, foreign DNA fragments from the surrounding environment.

• Transduction: Virus-mediated transfer where a bacteriophage carries bacterial DNA fragments from a donor to a recipient cell.

What critical condition distinguishes conjugation from transformation and transduction in experiment designs?

Conjugation strictly requires direct cell-to-cell contact. In classic "U-tube" filtration experiments, separating strains with a filter that prevents physical bacterial contact completely abolishes gene exchange via conjugation.

Define competent cells and transformants in the context of bacterial transformation.

• Competent cells: Bacteria that are physiologically capable of taking up foreign DNA from their environment.

• Transformants: Cells that have stably taken up the environmental DNA and integrated it into their own genome via recombination.

Describe the four distinct bacterial cell types involved in F factor conjugation based on their plasmid characteristics.

• F⁺ cell: Contains the F factor as a separate, autonomous circular plasmid; acts as a DNA donor.

• F⁻ cell: Lacks the F factor entirely; acts as a DNA recipient.

• Hfr cell: High Frequency Recombination cell; the F factor is integrated directly into the main bacterial chromosome.

• F' cell: Contains a separate circular plasmid that was excised from the chromosome but mistakenly carried some host bacterial genes with it.

What is the genetic outcome of an F⁺ × F⁻ cross?

Two F⁺ cells. The F⁻ recipient cell receives a copy of the F plasmid via single-strand transfer and replication, completely converting it into an F⁺ cell.

What is the genetic outcome of an Hfr × F⁻ cross and why?

One Hfr cell and one F⁻ cell (usually no change in recipient status). During transfer, the integrated F factor is nicked at oriT, moving chromosomal genes into the recipient. Because the connection usually breaks before the entire chromosome can transfer, the trailing portion of the F factor rarely makes it across, leaving the recipient F⁻.

What is the genetic outcome of an F' × F⁻ cross?

Two F' cells. The recipient cell receives the plasmid along with the specific host bacterial genes it carries, creating a partial diploid (merodiploid) for those specific genes.

What is a bacteriophage ?

A virus that infects and replicated within bacteria host cells

What happens to a host cell during the lytic cycle of a bacteriophage?

The phage binds to the bacterium, injects its DNA, digests the host bacterial DNA, replicates its own genome, assembles new viral proteins, and forces the host cell to lyse (burst) to release infectious progeny.

How does transduction accidentally transfer host bacterial genes?

During viral assembly in the lytic cycle, the bacterial chromosome is fragmented. The viral capsid mistakenly packages a piece of the host bacterial chromosome instead of viral DNA. When this defective phage infects a new recipient bacterium, it injects the donor bacterial genes, allowing them to recombine into the new host genome.

Lec 10 DNA and RNA

Who first isolated nucleic acid, and what was it originally called?

• Isolated in 1869 by Friedrich Miescher.

• He discovered it in the nuclei of white blood cells and named it nuclein. Because of its acidic properties, it later became known as nucleic acid.

What was Phoebus Levene's tetranucleotide theory, and how did it stall genetics research?

• Levene proposed around 1910 that DNA consisted of repeating, equal ratios of its four bases.

• This led scientists to incorrectly believe that DNA was too simple and uniform to carry complex genetic info. Consequently, for the next 30 years, it was widely assumed that proteins must be the genetic material because of their 20 variable amino acid subunits.

Describe Fred Griffith’s 1928 experiment and what concept it established.

• Griffith mixed live nonvirulent (Type IIR) bacteria with heat-killed virulent (Type IIS) Streptococcus pneumoniae.

• The mixture killed the mice, and live virulent bacteria were recovered.

• Conclusion: A chemical substance released from the dead bacteria had permanently changed the live strain, establishing the concept of the transforming principle.

How did Avery, MacLeod, and McCarty (1944) determine the exact chemical nature of the transforming principle?

• They treated virulent bacterial filtrates with specific enzymes: Protease (destroys proteins), RNase (destroys RNA), and DNase (destroys DNA).

• Only the culture treated with DNase failed to transform nonvirulent cells.

• Conclusion: The transforming principle is DNA.

Explain the Hershey-Chase (1952) experiment and how it confirmed DNA as the genetic material.

• They used T2 bacteriophages (50% protein / 50% DNA) to infect E. coli. They labeled phage protein coats with ³⁵S and phage DNA with ³²P.

• After blending and centrifuging to separate cell pellets from fluids, ³²P was recovered inside the bacterial pellet, while ³⁵S stayed in the fluid. Progeny phages were radioactive only when DNA was labeled.

• Conclusion:

  DNA—not protein—enters the cell and carries the genetic material.

What are the 4 different nitrogenous bases in DNA

adenine, cytosine, guanine, and thymine

What is a nucleosides?

A nitrogenous bases conjugated to sugars

What is a nucleotides? and where are things located on the sugar?

when a nitrogenous bases sugar is bonded to a phosphate group

the base is connected to the sugar at the 1’ carbon

the phosphate group is connected to the sugar at the 5’ carbon

What is the transforming principle?

An unknown substance that allowed harmless bacteria to permanently inherit the deadly characteristics of another bacterial stain

What is chargaff’s rules?

• Based on chemistry data across organisms, Erwin Chargaff showed that base compositions vary between species, but follow strict regularity:

  1. Adenine equals Thymine (\(A = T\)).

  2. Guanine equals Cytosine (\(G = C\)).

  3. The number of purines equals pyrimidines (\((A + G) = (T + C)\)).

What role did Rosalind Franklin and Maurice Wilkins play in solving DNA's structure, and what method did they use?

• They used X-ray diffraction, bombarding DNA crystals with X-rays to analyze atomic spacing from the resulting film pattern.

• Franklin’s photographs provided critical evidence that DNA is helical and has its sugar-phosphate backbone on the outside.

What two crucial conceptual leaps allowed Watson and Crick (1953) to successfully build the secondary structure model of DNA?

• 1. Base Pairing Chemistry: Realizing exactly how A pairs with T, and G with C (satisfying Chargaff's rules).

• 2. Antiparallel Strands: Recognizing that the two individual polynucleotide strands run in completely opposite structural directions.

How was it proven that RNA can also serve as genetic material, and by whom?

• Demonstrated in 1956 by Fraenkel-Conrad and Singer using the Tobacco Mosaic Virus (TMV) system.

• They created hybrid viruses mixing Type A RNA with Type B proteins (and vice-versa). The progeny viruses always matched the parent's RNA type, proving RNA carries the viral genome.

What are the three distinct chemical components that assemble into a single nucleotide?

1. A nitrogenous base.

2. A pentose (5-carbon) sugar.

3. A phosphate group (\(PO_{4}\), which yields nucleic acids' acidic nature).

Differentiate between the structures of purines and pyrimidines.

• Purines: Have a fused double-ring structure composed of 9 carbon/nitrogen atoms. Examples: Adenine (A) and Guanine (G).

• Pyrimidines: Have a single-ring structure composed of 6 carbon/nitrogen atoms. Examples: Cytosine (C), Thymine (T) (DNA), and Uracil (U) (RNA).

How do you distinguish a ribose sugar from a deoxyribose sugar by looking at its carbon ring numbering?

• Ribose (RNA): Features a reactive hydroxyl group (—OH) attached at the 2' carbon.

• Deoxyribose (DNA): Lacks an oxygen at the 2' carbon, possessing a simple hydrogen atom (—H) instead

To which specific carbon positions on the pentose sugar do the nitrogenous base and the phosphate group bind?

• The nitrogenous base links covalently to the 1' carbon.

• The phosphate group attaches to the 5' carbon.

What is the structural difference between a nucleoside and a nucleotide?

• Nucleoside: Consists strictly of a base conjugated to a sugar (e.g., deoxyadenosine).

• Nucleotide: A nucleoside that has added one or more phosphate groups covalently linked to its 5' carbon (e.g., deoxyadenosine 5'-monophosphate / dAMP).

What specific chemical bond connects individual nucleotides into a single polymer chain?

A phosphodiester linkage. This covalent bond links the 5'-phosphate group of an incoming nucleotide to the 3'-OH group of the preceding nucleotide.

How many hydrogen bonds form between the different complementary base pairs?

• T–A pairs: Joined by two hydrogen bonds.

• C–G pairs: Joined by three hydrogen bonds (making C-G bonds slightly stronger and harder to separate).

What is DNA helix and other major structure of B form DNA

Form a right handed double helix with a sugar phosphate backbone

winds in a way that it creates two unequal grooves

• one major: more accessible

• one minor less accessible

each 360 degree is around 3.4nm or 10base pairs

What’s the different between A form, B form and Z form of DNA

A form is when DNA is dehydrated so it is more wider and more compacted

B form is when DNA is hydrated

Z form is when DNA is left handed so spiraling the opposite direction

Map the directionality pathways of the Central Dogma vs. its Special Information Pathways.

• Central Dogma: Standard cellular flow travels from DNA Replication \(\rightarrow \) Transcription (DNA to RNA) \(\rightarrow \) Translation (RNA code to amino acid Protein sequence).

• Special Pathways: Chiefly found in viruses, where data travels via Reverse Transcription (RNA to DNA) or RNA Replication (RNA to RNA).

• What causes nucleic acids to form intra-molecular secondary structures like hairpins or stems?

• Occurs when a single-stranded molecule contains inverted repeats, allowing it to fold backward and hydrogen-bond to itself.

• Hairpin loops consist of a paired double-stranded stem and an unpaired single-stranded loop. RNA molecules frequently form highly intricate complex secondary structures because they lack a second external complementary strand.

What is DNA methylation, and which specific nucleotide base does it typically modify?

• A very common covalent modification where a methyl group (—CH₃) is attached directly to a base in genomic DNA.

• It most frequently targets Cytosine, transforming it into 5-Methylcytosine to regulate genomic function.

Lec 11 chromosome Organization and organelle genomes

What is DNA tertiary structure and why is it necessary?

• The higher-order three-dimensional organization of DNA in space.

• It is absolutely critical because a bacterial chromosome is about 1,000× the length of the cell, and human genomic DNA would stretch to about 6 feet long if placed end-to-end. Special packaging is required to fit this material inside the cell.

Differentiate between positive and negative supercoiling.

• Positive Supercoiling: Occurs when DNA is overwound (overrotated), adding turns to the helix and causing it to twist on itself.

• Negative Supercoiling: Occurs when DNA is underwound (underrotated), removing turns from

What do topoisomerases do?

Enzymes responsible for adding or removing supercoils from the relaxed DNA state. They accomplish this by breaking the nucleotide strands, rotating the ends of the helix to change the turn count, and then rejoining the strands together

What is a bacterial nucleoid?

The region in a prokaryotic cell where the bacterial chromosome organizes. It lacks a nuclear membrane and histones, consisting instead of a clump of twisted DNA loops held together by architectural proteins.

How is eukaryotic DNA is highly organized?

1. the doudle helix is wrap around histone prteins

Tight coiling into the chromatid of a chromosome

What is the different between euchromatin and heterochromatin?

• Euchromatin: Less condensed, located primarily on chromosome arms, contains unique sequences and many active genes. It replicates throughout the S phase, undergoes frequent transcription, and common crossing over.

• Heterochromatin: Highly condensed, located at centromeres, telomeres, and specific dense regions. It consists of repeated sequences with few genes, replicates late in the S phase, and exhibits infrequent transcription and uncommon crossing over (without active genes )

Describe the molecular structure of a nucleosome.

The most elementary unit of eukaryotic chromatin. It consists of a core of eight histone proteins (two copies each of H2A, H2B, H3, and H4) wrapped twice by DNA, separated by linker DNA.

Why is the centromere essential during cell division, and what are its sequence features?

• Function: It is the required organizing region that attaches to spindle microtubules during anaphase to move chromosome fragments to the spindle poles. Fragments lacking a centromere fail to attach and are lost from the nucleus, leading to degradation.

• Sequence Features: It contains no single conserved sequence consensus. Instead, it is characterized by repeated sequences, heterochromatin architecture, and specialized centromere-associated proteins.

What is the structure and function of a telomere?

• Stable caps found at the ends of eukaryotic linear chromosomes consisting of repeating sequences.

• They feature a long G-rich 3' single-stranded overhang that loops back and base-pairs natively with the telomeric double helix to lock into a protective structure called a t-loop. Telomeres shorten during somatic cell divisions because standard replication mechanisms cannot copy the extreme linear ends

Does genome size correlate directly with organismal complexity in eukaryotes?

No. Eukaryotic genome size varies massively and is a poor proxy for complexity. For example, the plant Arabidopsis thaliana has 125 million bp, humans (Homo sapiens) have ~3 billion bp, but corn (Zea mays) has 4.5 billion bp, and a salamander (Amphiuma) has an immense genome of 765 billion bp

Define DNA denaturation, annealing, and hybridization

• Denaturation (Melting): The complete separation of paired complementary strands by using heat or energy to break inter-base hydrogen bonds.

• Annealing (Renaturation): The structural reassembly of separated complementary strands as the solution cools and base-pair interactions reform.

• Hybridization: The annealing together of complementary single strands that originate from two completely different sources.

• What is melting temperature (T_m) and how does base composition alter it?

• (T_m) is the specific temperature at which a double-stranded DNA molecule denatures into single strands.

• Composition effect: A:T pairs possess only two hydrogen bonds, while G:C pairs share three hydrogen bonds. Therefore, G:C-rich sequences require more energy to break and possess a significantly higher (T_m) than A:T-rich sequences.

• What is the Endosymbiotic Theory and what genomic evidence supports it?

• The theory that mitochondria and chloroplasts originated 1.5 to 2 billion years ago when an anaerobic ancestral eukaryote engulfed an aerobic \(\alpha \)-proteobacterium (and later a photosynthetic cyanobacterium) via endocytosis.

• Genomic Evidence: Both organelles retain their own distinct, circular genomes inside the cell matrix/stroma, copy numbers vastly outnumber nuclear genomes, and they maintain their own protein synthesis machinery (encoding their own distinct rRNAs and tRNAs).

How did Meselson and Stahl (1958) prove that DNA replication is semiconservative?

• They labeled E. coli with heavy (¹⁵N) isotopes, then transferred them to light (¹⁴N) medium.

• Round 1: Produced a single intermediate-weight DNA band (ruling out the conservative model, which would predict separate heavy and light bands).

• Round 2: Produced two clear bands—one light (¹⁴N) and one intermediate. Because the pure (¹⁵N) heavy band never reappeared and intermediate hybrid DNA persisted, it proved replication is semiconservative (each original strand serves as a permanent template for a new strand).

conservative vs dispersive vs semiconservative replication

semiconservative replication- one strand serve as a template meaning one the old and one is new

conservative: make a new structurc witout taking from the DNA

Explain the mechanics and features of the Theta (0)) replication model.

• Template: Circular DNA (common in bacteria like E. coli).

• Mechanics: No strand breakage occurs. DNA unwinds at a single origin of replication, creating a single replication bubble with a replication fork at each end. Synthesis proceeds unidirectionally or bidirectionally around the circle until two circular molecules are produced.

Explain the mechanics and features of Rolling-Circle replication.

• Template: Circular plasmids and viral genomes.

• Mechanics: Requires a physical break (nick) in one of the nucleotide strands. The free 3'-OH end of the broken strand is elongated, using the unbroken inner strand as a template. This progressively displaces the 5' end of the broken strand as a single-stranded linear tail.

• Products: One circular molecule and one linear molecule (the linear tail can circularize to synthesize a complementary strand). Only uses 1 replicon and is unidirectional.

Explain the mechanics and features of Linear Eukaryotic replication.

• Template: Large linear eukaryotic chromosomes.

• Mechanics: No strand breakage occurs. It utilizes hundreds to thousands of replication origins across the chromosome. Each origin forms a replication bubble with bidirectional forks that migrate outward. Adjacent bubbles run into each other, and the newly synthesized segments fuse together.

• Products: Two identical linear DNA molecules.

• What is a replicon, and why do eukaryotes require thousands of them?

• A unit of DNA operating under the control of a single origin of replication.

• E. coli has a single replicon spanning its entire 4.6 million bp genome. Eukaryotes possess much larger genomes and slower replication rates; utilizing thousands of smaller, concurrent replicons (averaging 20,000–300,000 bp) compresses the time required to copy the total genome from many days down to just a few hours.

lec 12

What are the three fundamental components strictly required for DNA replication to occur?

1. A single stranded template to guide nucleotide selection

2. Building blocks to synthesize the new strand

3. Enzymes and accessory proteins to perform the physical work of synthesis and unwinding

What concrete building blocks are used to extend a growing DNA strand, and where does the energy for linkage originate?

• dNTPs (deoxyribonucleoside triphosphates) are the essential building blocks.

• The raw chemical energy required to form the covalent phosphodiester bond is stored inside the high-energy triphosphate bonds of the dNTP itself. When a nucleotide is incorporated, two phosphate groups are cleaved off (as pyrophosphate), releasing the energy needed to drive the reaction.

• Why must DNA synthesis strictly proceed in a 5' \(\rightarrow \) 3' direction?

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• Because DNA polymerases require a free 3'-hydroxyl (—OH) group on the preceding nucleotide. This 3'-OH group executes a nucleophilic attack on the 5'-phosphate group of the incoming dNTP substrate. Consequently, the complementary template strand is always read in a 3' \(\rightarrow \) 5' direction.

Explain how the antiparallel nature of DNA template strands forces a divergent direction of synthesis at a single replication fork

Because the two template strands are antiparallel, one template exposes a 3' \(\rightarrow \) 5' orientation toward the fork while the other exposes a 5' \(\rightarrow \) 3' orientation. Since synthesis must run 5' \(\rightarrow \) 3', DNA polymerases must move in chemically opposite directions on the two strands: one replicates continuously toward the unwinding fork, while the other replicates discontinuously away from it

Compare the structural mechanics of leading strand synthesis vs. lagging strand synthesis

• Leading Strand: Undergoes continuous replication. Its polymerase moves in the exact same direction as the unwinding replication fork, meaning it can continuously grow down the template as new single-stranded DNA is exposed.

• Lagging Strand: Undergoes discontinuous replication. Its polymerase moves in the direction opposite to that of unwinding. As the fork opens, synthesis must periodically restart further back at the fork, moving away from it until it runs out of template

What are Okazaki fragments, and why are they formed

Short fragments of newly synthesized DNA produced by discontinuous synthesis on the lagging strand. They are formed because DNA polymerase can only build in a 5' \(\rightarrow \) 3' direction away from the replication fork, requiring synthesis to be re-initiated at periodic intervals by a primer as the fork continues to unwind

How do the leading and lagging strands balance out concurrently within the Theta (\(\theta \)) and Linear Eukaryotic models vs. the Rolling-Circle mode

• Theta & Linear Models: Unwinding at the origin creates a bubble with two distinct replication forks moving away from each other. As a result, each fork contains one leading strand and one lagging strand operating simultaneously.

• Rolling-Circle Model: Utilizes a single nicked strand and a single direction of unwinding, meaning only continuous leading strand synthesis occurs off the circular template initially.

Map the precise step-by-step sequence of events that occurs when bacterial replication is initiated at oriC

1. Specialized initiator proteins (DnaA) bind directly to the oriC locus.

2. This binding forces a short stretch of local DNA to unwind, creating an initiation bubble.

3. The opening allows DNA helicase enzymes and single-strand-binding proteins to safely attach to the single-stranded DNA templates.

What is the precise function of DNA helicase, and what thermodynamic cost does it pay

• Function: It binds to the lagging-strand template at each replication fork and travels in a 5' \(\rightarrow \) 3' direction, physically moving the replication fork forward.

• Thermodynamic Cost: It must actively expend chemical energy (ATP hydrolysis) to break the hydrogen bonds holding the complementary nitrogenous base pairs together, separating double-stranded DNA into single strands.

What are single-strand-binding proteins (SSBs) and why are they necessary

Proteins that selectively bind to the exposed single-stranded DNA template directly behind the helicase. They are required to stabilize and protect the single-stranded DNA, preventing it from snapping back into a double helix or folding into aberrant secondary structures before polymerase can replicate it.

What is DNA gyrase, and what severe biophysical hazard does it resolve during replication

• A specialized topoisomerase that relieves the severe torsional strain (supercoiling) that accumulates ahead of the replication fork as DNA helicase forces the double helix to unwind.

• Mechanism: It cuts the double-stranded backbone, rotates the ends around each other to undo the supercoils, and then seals the strands back together.

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What is the fundamental biological limitation of DNA Polymerase, and how does primase rescue it

• Limitation: DNA polymerase is completely incapable of initiating DNA synthesis from scratch on a raw single strand; it can only append a nucleotide to a pre-existing 3'-OH group.

• Rescue: Primase (an RNA polymerase) synthesizes a short stretch of complementary RNA nucleotides (a primer). This provides the mandatory initial 3'-OH group that DNA polymerase can dock onto to begin elongation

Compare the primer requirements of the leading strand vs. the lagging strand

• The leading strand requires only a single RNA primer at the absolute 5' end of the newly synthesized strand, because its elongation is continuous down the template.

• The lagging strand requires a brand-new primer to be generated by primase at the beginning of every single Okazaki fragment

What is the primary elongation enzyme in E. coli, and how does it maintain hyper-accuracy

• DNA Polymerase III is the large multi-protein complex responsible for the bulk of bacterial elongation.

• Accuracy: It maintains high fidelity via a built-in 3' \(\rightarrow \) 5' exonuclease activity called proofreading. If an incorrect nucleotide is accidentally paired, the enzyme detects the structural distortion, stalls replication, backtracks to excise the inaccurate base, and replaces it with the correct dNTP before moving forward

Explain how RNA primers are systematically removed and replaced with DNA in prokaryotes

When DNA Polymerase III reaches an downstream RNA primer, it stops. DNA Polymerase I takes over, utilizing the 3'-OH group of the upstream DNA fragment as a starting point. Moving 5' \(\rightarrow \) 3', it systematically displaces and degrades the RNA primer nucleotides and fills the resulting gap with accurate DNA nucleotides

• What is a backbone nick, and what enzyme seals it?

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• A nick is a single missing phosphodiester bond in the sugar-phosphate backbone between adjacent nucleotides, left behind after DNA Polymerase I finishes replacing an RNA primer with DNA.

• DNA Ligase seals this nick by covalently bonding the 5'-phosphate group of the first nucleotide added by DNA Polymerase III to the 3'-OH group of the final nucleotide added by DNA Polymerase i

• What unique structural challenge must eukaryotic replication machinery navigate immediately after synthesis that bacteria bypass?

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Eukaryotes must manage nucleosomes. Eukaryotic cells maintain highly specialized chromatin-remodeling mechanisms that systematically re-assemble and re-position nucleosome histone octamers back onto the newly synthesized double-stranded regions immediately behind the progressing replication fork

• Explain the end-replication problem in linear chromosomes.

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• In linear DNA, when the terminal RNA primer on the lagging strand's extreme end is removed by a polymerase, there is no upstream 3'-OH group available to serve as a substrate for DNA polymerase to fill the gap. Consequently, DNA replication leaves an unreplicated gap at the end of the chromosome, causing the chromosome to shorten progressively with every round of cell division.

• What is telomerase, and how does it exploit an internal template to preserve chromosome ends?

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• An enzyme complex composed of protein and an internal RNA template that functions to extend linear chromosome caps (telomeres).

• Mechanism: The RNA portion of telomerase base-pairs with the G-rich terminal single-stranded 3' overhang of the telomere. Telomerase uses its own RNA loop as a template to synthesize and append new DNA repeats to the 3' end. Once the overhang is elongated, normal primase and DNA polymerase can lay down a primer and synthesize a complementary strand, successfully filling the end gap

Describe the cellular distribution of telomerase activity and its connection to cellular aging

• Telomerase activity is highly active in embryonic cells, germline gametes, and certain highly proliferative stem cells, but is completely absent from most somatic cells. Over time, as somatic cells divide, their telomeres progressively shorten. Once telomeres reach a critically short threshold, the chromosomes become structurally unstable, triggering programmed cell death (apoptosis).

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Initiator Protein (DnaA)

Binds to oriC to cause a short stretch of localized double helix to unwind

DNA Helicase

Consumes energy to break hydrogen bonds, separating DNA into single strands at the fork.

Single-Strand-Binding (SSB)

Binds and stabilizes single-stranded template DNA, protecting it from re-annealing.

DNA Gyrase (Topoisomerase)

Cuts and reseals DNA ahead of the fork to release accumulated torsional supercoiling strain.

Primase

Synthesizes a short complementary RNA primer to supply a mandatory free 3'-OH group.

DNA Polymerase III

Elongates the new strand 5→ 3' and proofreads mistakes via 3' → 5' exonuclease activity.

DNA Polymerase I

Removes the RNA primer nucleotides and fills the resulting gap with DNA nucleotides

DNA Ligase

Seals the single-strand nick in the sugar-phosphate backbone with a phosphodiester bond.

Lec 13 Transcription

What are the similarities and differences of RNA vs DNA

Similarities are:

• both share the Same 5’ to 3’ linkage

• composed of nucleotides

• nucleotides joined by phosphodiester

Differences are:

• RNA is single stranded while DNA is double

• RNA contains uracil bases while DNA contain thymine

• RNA has hydroxyl groups at the 2’ position of the ribose sugar (making is more reactive and less stable) while DNA has a H group on the 2’ position of the deoxyribose

• RNA molecule folds to secondary structure through self base pairing making many types while DNA is double helix

What is transcription?

When RNA is generated from a DNA template

Durring reading DNA is 3’ to 5’ direction while RNA synthesis to a 5’ to 3’ direction

the 3’OH of the ribose sugar at the end of the chain is used to add new nucleotides

What are the different forms of RNA transcripts that can transcribed in both prokaryotic and eukaryotic cells?

• mRNA = messenger, encode protein sequences (nucleus and cytoplasm)

• tRNA= transfer, help in protein synthesis by adding amino acids into polypeptide chain (cytoplasm)

• rRNA = ribosomal, help in protein synthesis by structural and functional components (cytoplasm)

What are the different forms of RNA transcripts that can transcribed only in eukaryotic cells

snRNA (small nuclear RNA) are involved in splicing while snoRNA (small nucleolar RNA) are involved in processing of rRNA

miRNA (microRNA) and siRNA (small interfering RNA) are short RNAs that can have major effects on gene expression

What is a promoter?

the site upstream of the direction of transcription, it defines where a transcript begins and the direction it will be transcribed

What is a terminator?

downstream of transcription and determines the place where the

transcript will end