Bio 151 - Exam 2 (Genetics)

Darwin & Wallace's Theory of Natural Selection

The theory that individuals with heritable traits better suited to their environment survive and reproduce more successfully, passing those traits to offspring — the mechanism driving evolutionary change over generations.

Mendel's Laws of Inheritance

Two principles governing how traits are passed from parents to offspring: (1) Law of Segregation — two alleles for a gene separate during gamete formation; (2) Law of Independent Assortment — alleles of different genes on different chromosomes sort independently into gametes.

The Structure of DNA

DNA is a right-handed antiparallel double helix composed of two polynucleotide strands. The sugar-phosphate backbone runs on the outside; complementary bases (A-T, G-C) pair inward and are held together by hydrogen bonds and base-stacking interactions.

Nucleotides

The monomeric building blocks of DNA (and RNA), each consisting of a 5-carbon sugar (deoxyribose in DNA, ribose in RNA), a phosphate group, and a nitrogenous base. Nucleotides are linked by phosphodiester bonds to form polynucleotide chains.

Nucleosides

A nucleotide without the phosphate group — just a nitrogenous base covalently attached to a 5-carbon sugar (deoxyribose or ribose). Example: adenosine = adenine + ribose.

Phosphodiester Bonds

The covalent bonds that link nucleotides together in a DNA or RNA strand. Formed between the 3'-OH of one sugar and the 5'-phosphate of the next nucleotide, creating the sugar-phosphate backbone.

The Double Helix

The three-dimensional structure of DNA — two antiparallel polynucleotide strands wound around each other in a right-handed helix. Proposed by Watson and Crick in 1953 based on data from Rosalind Franklin and Chargaff.

The Pairing of Nucleotide Bases

Complementary base pairing between the two strands of DNA: Adenine (A) pairs with Thymine (T) via 2 hydrogen bonds; Guanine (G) pairs with Cytosine (C) via 3 hydrogen bonds. In RNA, U (uracil) replaces T.

Antiparallel DNA Strands

The two strands of DNA run in opposite directions: one strand runs 5'→3' and the other runs 3'→5'. This antiparallel orientation is essential for proper base pairing and is dictated by the phosphodiester bond chemistry.

Explain the experiments that indicated that the material of heredity is DNA rather than proteins.

(1) Griffith (1928): Heat-killed virulent (smooth) S. pneumoniae transformed live non-virulent (rough) bacteria into virulent ones — a 'transforming principle' existed. (2) Avery, MacLeod & McCarty (1944): Showed the transforming principle was DNA — DNase abolished transformation; protease did not. (3) Hershey-Chase (1952): Phage T2 labeled with ³²P (DNA) or ³IS (protein). Only ³²P entered bacteria and was inherited by progeny phage — DNA is the hereditary material.

Describe the components of DNA.

DNA is a polymer of nucleotides. Each nucleotide: (1) 5-carbon deoxyribose sugar, (2) phosphate group at 5' carbon, (3) nitrogenous base at 1' carbon (purines: A, G; pyrimidines: C, T). A nucleoside = sugar + base only. Nucleotides linked by phosphodiester bonds (3'-OH of one sugar → 5'-phosphate of next) form the backbone.

Describe the types of data that contributed to understanding the structure of DNA, such as data from Rosalind Franklin.

(1) Chargaff's Rules (1950): [A]=[T] and [G]=[C] — implied base pairing. (2) Franklin's X-ray crystallography (Photo 51, 1952): revealed helical structure, 2 nm diameter, 0.34 nm/bp, 3.4 nm/full turn (10 bp/turn). (3) Watson & Crick (1953): combined these data to build the antiparallel double helix model.

Describe the structure of DNA, including the bonds that connect the components.

Right-handed antiparallel double helix. Sugar-phosphate backbone outside; bases inside. Covalent phosphodiester bonds link nucleotides within each strand. Hydrogen bonds hold strands together: A-T (2 H-bonds), G-C (3 H-bonds). Hydrophobic base-stacking interactions further stabilize the helix.

The Central Dogma

The flow of genetic information in cells: DNA → RNA → Protein. DNA is transcribed into mRNA, which is translated into protein. Reverse transcriptase (in retroviruses) allows RNA → DNA, but proteins are never reverse-translated into nucleic acid.

DNA Replication (Semiconservative)

The mechanism of DNA copying in which each new double-stranded DNA molecule consists of one original (parental) strand and one newly synthesized strand. Demonstrated by the Meselson-Stahl experiment using ¹IN/¹IN density labeling.

Helicase

An enzyme that unwinds and separates the two strands of the DNA double helix at the replication fork by breaking the hydrogen bonds between base pairs, powered by ATP hydrolysis.

Single-Strand Binding (SSB) Proteins

Proteins that bind and stabilize the single-stranded DNA template strands after helicase unwinds them, preventing them from re-annealing or forming secondary structures during replication.

Topoisomerase

An enzyme that relieves the torsional stress (supercoiling) that builds up ahead of the replication fork as helicase unwinds DNA. Type I cuts one strand; Type II cuts both strands, passes a segment through, and reseals.

DNA Polymerase

The enzyme that synthesizes new DNA strands by adding dNTPs to the 3'-OH end of a primer, reading the template 3'→5' and synthesizing 5'→3'. Has proofreading (3'→5' exonuclease) activity. Cannot initiate new strands — requires a primer.

RNA Primase

A specialized RNA polymerase that synthesizes short RNA primers (~10 nucleotides) complementary to the DNA template. Provides the free 3'-OH group that DNA polymerase needs to begin DNA synthesis.

RNA Primer

A short RNA sequence (~10 nt) synthesized by primase that is complementary to the DNA template and provides a free 3'-OH for DNA polymerase to extend. Primers are later removed and replaced with DNA.

Ligase

DNA ligase is the enzyme that seals the nicks (breaks in the phosphodiester backbone) between adjacent Okazaki fragments on the lagging strand, forming a continuous strand. Uses NAD+ or ATP as energy source.

Leading Strand

The daughter strand synthesized continuously in the 5'→3' direction toward the replication fork, following the movement of helicase. Requires only one primer.

Lagging Strand

The daughter strand synthesized discontinuously away from the replication fork in short Okazaki fragments (~100-200 nt in eukaryotes), each requiring its own primer. Ultimately joined by ligase.

Okazaki Fragments

Short segments of DNA (~1,000-2,000 nt in prokaryotes; ~100-200 nt in eukaryotes) synthesized discontinuously on the lagging strand template. Named after Reiji Okazaki. Joined together by DNA ligase after primer removal.

Telomere

Repetitive non-coding DNA sequences (TTAGGG in humans) at the ends of linear chromosomes that protect coding sequences from the end-replication problem. Shorten with each cell division in most somatic cells.

Telomerase

A reverse transcriptase enzyme that extends the telomere by using its own internal RNA template to add telomeric repeat sequences to chromosome ends. Active in germ cells, stem cells, and most cancer cells (~90%).

Describe the process of DNA Replication.

Semiconservative replication proceeds in three stages: (1) Initiation: origins of replication (ori) are recognized; helicase opens the double helix forming a replication bubble with two bidirectional forks. (2) Elongation: primase lays RNA primers; DNA Pol III extends them 5'→3'. Leading strand = continuous; lagging strand = Okazaki fragments. DNA Pol I removes primers and fills gaps; ligase seals nicks. (3) Termination: forks meet; topoisomerase resolves interlinked molecules.

Complementary Nucleotides

Nucleotides on opposite DNA strands match through hydrogen bonding between their nitrogenous bases.

A pairs with T

A pairs with T via 2 hydrogen bonds.

G pairs with C

G pairs with C via 3 hydrogen bonds.

Specificity of Base Pairing

Specificity comes from the geometry and position of hydrogen bond donors/acceptors.

G-C Pairs Stability

G-C pairs are more thermally stable.

Complementarity

One strand's sequence dictates the other's.

Predicting Nucleotide Sequence

Read the template 3'→5' and write the new strand 5'→3', substituting A↔T and G↔C.

Example of Nucleotide Prediction

Template 3'-ATCGGCTA-5' → New strand 5'-TAGCCGAT-3'.

SSB Proteins

Single-stranded binding proteins that stabilize ssDNA.

Primase

Enzyme that makes RNA primers.

DNA Pol III

Enzyme that replicates DNA 5'→3' with proofreading.

DNA Pol I

Enzyme that removes primers and fills gaps.

Sliding Clamp (PCNA)

Protein that holds DNA polymerase on the template.

DNA Polymerase Addition

DNA Pol adds dNTPs to the 3'-OH of the growing strand.

NTP Addition Mechanism

The 3'-OH attacks the alpha-phosphate of the incoming dNTP.

Pyrophosphate Release

Pyrophosphate (PPi) is released and hydrolyzed to 2 Pi, driving the reaction.

DNA Pol Starting Requirement

DNA Pol cannot start de novo — requires a 3'-OH from a primer.

Primers in DNA Replication

Primers are needed because DNA Pol cannot start a new strand.

Primase Function

Primase synthesizes a short RNA primer (~10 nt) complementary to the template.

Mistakes in DNA Replication

DNA Pol has 3'→5' exonuclease proofreading to detect mismatches.

Error Rate

Error rate is approximately ~1/10^10.

Mismatch Repair Proteins

Proteins (MutS, MutL, MutH) that catch remaining errors.

DNA Replication Start

Initiator proteins recognize ori, recruit helicase, open helix to form a replication bubble.

DNA Replication End

Forks meet; prokaryotes use topoisomerase IV to decatenate.

PCR

PCR amplifies a specific DNA sequence in vitro.

PCR Cycle Steps

1. Denaturation ~95°C, 2. Annealing ~50-65°C, 3. Extension ~72°C.

Telomere Shortening

The 'end-replication problem' leads to chromosome shortening ~50-100 bp per division.

Telomere Repair

Telomerase binds the 3' overhang and adds TTAGGG repeats.

Differences between DNA and RNA

DNA: deoxyribose, double-stranded, thymine; RNA: ribose, single-stranded, uracil.

Transcription

The process by which RNA polymerase synthesizes RNA from a DNA template.

Transcription Initiation

Stage when RNA polymerase recognizes and binds the promoter.

Transcription Elongation

Stage during which RNA polymerase adds ribonucleotides to the growing RNA chain.

Promoter

A DNA sequence upstream of a gene where RNA polymerase binds to initiate transcription.

Terminator

A DNA sequence that signals the end of transcription.

Transcription Factors (TFs)

Proteins that regulate the initiation of transcription by binding to DNA sequences.

Enhancer Sequence

A cis-regulatory DNA element that increases transcription when bound by activator proteins.

Transcription Activator Protein

A transcription factor that binds to enhancer sequences and stimulates transcription.

Mediator Complex of Proteins

A multi-subunit protein complex that acts as a bridge between activator proteins and the transcription machinery.

RNA Polymerase

The enzyme that synthesizes RNA from a DNA template without requiring a primer.

5' Cap (RNA Processing)

A 7-methylguanosine cap added to the 5' end of eukaryotic pre-mRNA.

Polyadenylation

The addition of a poly(A) tail to the 3' end of eukaryotic pre-mRNA.

RNA Splicing

The removal of introns and joining of exons from the pre-mRNA transcript.

Intron

A non-coding intervening sequence within a eukaryotic pre-mRNA that is removed during splicing.

Exon

A coding sequence in a eukaryotic gene that is retained in the mature mRNA after splicing.

Alternative Splicing

A process that produces multiple distinct mature mRNA isoforms from the same pre-mRNA.

Exons

Translated into protein sequence. The term can also refer to non-translated but retained sequences (5' UTR, 3' UTR).

Purpose of Transcription

Produce portable RNA (mRNA, rRNA, tRNA, etc.) for protein synthesis or direct function.

Stages of Transcription

Three stages: Initiation (RNA Pol binds promoter, opens DNA), Elongation (RNA synthesized 5'→3'), Termination (RNA Pol released at terminator sequence).

Enzymes and Proteins in Transcription

RNA Pol: core enzyme — reads template 3'→5', synthesizes RNA 5'→3', no primer needed, no proofreading. Sigma (prokaryotes): promoter recognition. GTFs (eukaryotes): TFIID/TBP binds TATA box; TFIIH unwinds DNA and phosphorylates CTD. Activators: bind enhancers → stimulate via Mediator complex.

Initiation of Transcription

Prokaryotes: sigma guides RNA Pol to -10/-35 promoter; DNA melts → open complex. Eukaryotes: GTFs assemble at TATA box; TFIIH unwinds DNA and phosphorylates CTD of RNA Pol II → promoter clearance. Enhancers loop to promoter via Mediator to boost transcription.

RNA Synthesis

RNA Pol reads template 3'→5', adds complementary rNTPs 5'→3' (A→U, T→A, G→C, C→G). No primer needed. Transcription bubble (~12-14 nt) moves with Pol. No proofreading → error rate ~1/10I-10I.

Differences between DNA Replication and RNA Transcription

Primer: DNA Pol needs one; RNA Pol does not. Product: DNA vs. RNA. Nucleotides: dNTPs vs. rNTPs (ribose + U). Scope: entire genome vs. individual genes. Proofreading: DNA Pol yes; RNA Pol no. Both synthesize 5'→3'.

Processing of Primary RNA Transcript

(1) 5' Capping: mIG cap added co-transcriptionally → protects, aids export and ribosome recognition. (2) 3' Polyadenylation: cleavage at AAUAAA signal; ~200 A residues added → stability and export. (3) Splicing: spliceosome removes introns, joins exons. Alternative splicing → multiple protein isoforms from one gene.

Translation

The process by which the ribosome decodes the mRNA sequence (in codons) to synthesize a polypeptide chain.

Initiation (Translation)

The first stage of translation in which the ribosomal subunits, initiator tRNA, and mRNA assemble at the AUG start codon.

Elongation (Translation)

The repetitive stage of translation in which the ribosome moves along the mRNA one codon at a time, adding amino acids to the growing polypeptide chain.

Termination (Translation)

The final stage of translation triggered when a stop codon (UAA, UAG, UGA) enters the A site.

Ribosomes

Large ribonucleoprotein complexes (rRNA + proteins) that are the molecular machines of translation.

Transfer RNA (tRNA)

Adapter RNA molecules (~75-95 nt) that carry specific amino acids to the ribosome.

Aminoacyl-tRNA Synthetases

A family of enzymes (one per amino acid) that catalyze the covalent attachment of the correct amino acid to its cognate tRNA, using ATP.

E, P, A Sites on the Ribosome

The three tRNA binding sites: A (aminoacyl) site — accepts incoming aminoacyl-tRNA; P (peptidyl) site — holds the tRNA bearing the growing polypeptide chain; E (exit) site — holds the deacylated tRNA before it exits the ribosome.

Codon

A sequence of three nucleotides (triplet) in mRNA that specifies a particular amino acid or a stop signal.

AUG Codon

The universal start codon in mRNA that encodes methionine (Met) in eukaryotes or formyl-methionine (fMet) in prokaryotes.

Stop Codon

One of three mRNA codons (UAA, UAG, UGA) that signal the end of translation.

Release Factors

Proteins that recognize stop codons in the A site of the ribosome and promote hydrolysis of the bond between the polypeptide and the P-site tRNA.

Polycistronic mRNA

An mRNA that contains multiple open reading frames (ORFs) encoding more than one protein, each with its own start and stop codon.

Purpose of Translation

Convert nucleotide language (codons) into amino acid language (protein).

Steps involved in Initiation of Translation

Prokaryotes: 30S binds Shine-Dalgarno sequence; fMet-tRNA binds AUG in P site; 50S joins → 70S complex. Eukaryotes: 43S complex (40S + eIF2-GTP + Met-tRNA) scans from 5' cap → finds AUG (Kozak); 60S joins → 80S.

Steps involved in Elongation of Translation

(1) Decoding: EF-Tu/eEF1A delivers correct aminoacyl-tRNA to A site; (2) Peptide bond formation: peptidyl transferase (rRNA ribozyme) transfers growing peptide to A-site amino acid; (3) Translocation: EF-G/eEF2 moves ribosome 3 nt.

Steps involved in Termination of Translation

Stop codon (UAA, UAG, UGA) enters A site. Release factors (RF1/RF2 prokaryotes; eRF1 eukaryotes) bind A site → hydrolyze peptidyl-tRNA bond → polypeptide released.

Differences between Prokaryotic and Eukaryotic Translation

Ribosomes: 70S (30S+50S) vs. 80S (40S+60S). Initiation: Shine-Dalgarno + fMet-tRNA vs. 5' cap scanning + Kozak + Met-tRNA. Coupling: prokaryotes can translate co-transcriptionally.

Mutation

A heritable change in the DNA sequence of an organism.