AP Biology: DNA, RNA, and Gene Expression

• This video is designed to help students prepare for the AP Biology exam and unit 6 test
• The video will cover complex material related to RNA production, modification, and translation into protein
• The video will cover DNA and RNA structure and function
• DNA replication
• Transcription
• Translation
• The genetic code
• Regulation of gene expression in prokaryotes (operons)
• Eukaryotic gene expression
• Mutation and horizontal gene transfer
• Biotechnology
• The video is presented by Glenn Wolkenfeld (Mr. W), a retired AP biology teacher
• There is a downloadable checklist available at AP bios to aid study

DNA Structure

• DNA consists of two strands arranged in a double helix.
• Nucleotide monomers compose each strand.
• Deoxyribose, a phosphate group, and a nitrogenous base make up a nucleotide.
• Adenine, thymine, guanine, and cytosine are the four possible nitrogenous bases.
• The sugar-phosphate backbone consists of deoxyribose sugars and phosphate groups linked by covalent bonds.
• Bases with complementary shapes bind via hydrogen bonds, adenine with thymine and guanine with cytosine.
• The binding of nucleotides requires them to be oriented upside down relative to each other, making the strands antiparallel.

DNA as Hereditary Molecule

• Four bases in any order provide information storage
• The sequence isn't determined by DNA's chemistry, allowing it to be an informational code that specifies RNA and protein sequences.
• The specific base pairing (A-T, G-C) enables each strand to template the synthesis of a complementary strand during DNA replication.
• Accurate transmission of genetic information is ensured from parent cells to daughter cells.
• Double helical structure protects base sequence while allowing mutation
• Bases changing spontaneously/due to environmental factors causes a low level of mutation
• Mutation allows code change and evolution

DNA vs. RNA Functions

• DNA is the molecule of heredity in all cell-based life.
• RNA serves as the hereditary molecule in some viruses (e.g., HIV, SARS-CoV-2).
• RNA participates in information transfer related to protein synthesis (mRNA, tRNA, rRNA) in all organisms.
• RNA regulates gene expression in eukaryotes through splicing introns from pre-mRNA and controlling protein synthesis.

Information Storage: Prokaryotes vs. Eukaryotes

• Prokaryotes store DNA in looped circular chromosomes.
• Bacterial and archaeal genomes range from around a 100,000 base pairs to 10 million base pairs.
• Proteins do not wrap around prokaryotic DNA.
• Eukaryotic DNA is arranged into multiple linear chromosomes.
• Histones, structural proteins, wrap eukaryotic DNA.
• The human genome contains 3.2 billion base pairs, whereas some plant genomes contain 150 billion base pairs.

Plasmids

• Plasmids are small extra-chromosomal DNA loops.
• Plasmids are commonly found in bacteria, less so in archaea, and rarely in eukaryotes.
• Horizontal gene transfer between bacterial cells relies on them.
• Plasmids often carry antibiotic resistance genes.
• Plasmids are used in genetic engineering for DNA replication and expressing engineered genes in bacteria.

DNA Replication

• DNA replication uses enzymes to synthesize daughter strands, using each strand of the double helix as a template
• Double helix separates into single strands, DNA polymerase binds nucleotides via base pairing rules to the template
• Each daughter DNA double helix consists of one conserved parent molecule strand, and one newly synthesized strand.
• Semiconservative replication means one strand is conserved whereas the other one is new

DNA Replication: Starting the Process

• Helicase separates the double-stranded DNA at the origin of replication.
• Separating double-stranded DNA involves breaking the hydrogen bonds, which holds the strands together.
• The replication fork is a structure that is created by the exposure of the single strand.

DNA Replication: Key Enzymes

• Primase is what lays down the RNA primers
• DNA polymerase binds new nucleotides to the three prime end of a growing strand creating a sugar phosphate bond.
• DNA polymerase requires an RNA primer to start connecting DNA nucleotides to, as it can only add nucleotides to an existing strand.
• Single-strand binding proteins prevent the double helix from rewinding, allowing other enzymes to carry out replication.

DNA Replication: Leading vs. Lagging Strand

• DNA replication is continuous on the leading strand, where DNA polymerase follows the opening replication fork.
• Polymerase synthesizes in the opposite direction from the opening replication fork from the lagging strand
• The lagging strand synthesizes built from short Okazaki fragments, which makes replication discontinuous.

DNA Replication: Finishing the Process

• To remove RNA primers and replace with DNA, DNA polymerase 1 is used.
• DNA ligase seals gaps between fragments to create complete daughter strands, by using sugar phosphate bonds

Transcription

• Transcription is the creation of RNA from DNA.
• The DNA starts with a promoter region which indicates the start of genes
• An enzyme called RNA polymerase binds with the promoter dna.
• RNA Polymerase transcribes the sequence of DNA bases on DNA's template strand into a sequence of RNA
• DNA is read in the three prime to five prime direction, and RNA is synthesized in the five prime to three prime direction for all enzymes working with DNA
• Transcription ends when the RNA polymerase reaches a Terminator region and separates from the DNA

Transcription: Template vs. Coding Strand

• Transcribed from DNA to RNA, the template strand is also called the noncoding, antisense, or minus strand.
• Coding strand is complementary to the template strand and has the same nucleotide sequence as the RNA

Transcription: Prokaryotes

• Because there is no separation between genetic material and the cytoplasm, procaryotes do not have a nucleus
• Therefore, transcribed RNA can immediately be translated by ribosomes into protein
• Polysomes are multiple ribosomes that read the same RNA strand.

Genetic Code

• Living things translate nucleotide sequences into amino acid sequences using the genetic code.
• Every three RNA nucleotide groups (codons) code for one amino acid.
• The code is used by nearly every living thing
• Every codon can determine one amino acid
• There are synonymous codons

Translation

• There are different roles that participants play in translation
• mRNA contains codons which specify the order of amino acids
• Ribosomes connect amino acids to create a polypeptide
• TRNAs bring amino acids to bring to the ribosome
• TRNAs have an anticodon and an amino acid binding site

Ribosomes

• Ribosomes translate information from mRNA into amino acid sequences.
• Polypeptides are formed from amino acids linked together by ribosomes
• They have a large and a small subunit and three TRNA binding sites, E, P, and A which stand for exit, polypeptide, and aminoacyl respectively

Translation: Initiation

• Processed mRNA leaves the nucleus through a nuclear pore
• The small ribosomal subunit binds with mRNA and goes to the start codon AUG, where translation begins
• The small subunit then Waits for a TRNA with matching anticodon to bind with the start codon
• The large subunit binds with the small subunit and makes the ribosome complete
• The first TRNA with methionine is at the p site

Translation: Elongation

• The next TRNA goes to the asite and carries a new amino acid
• A peptide bond forms between the p and asite amino acids catalyzed by the ribosome
• The ribosome moves over one codon during translocation
• A dipeptide hangs off of the psite amino acid, whereas the asite is empty
• The process continues through another round of translocation that creates a tripeptide, and occurs along the entire mRNA length

Translation: Termination

• The ribosome reaches a stop codon that has no corresponding TRNA
• Instead a release factor protein binds with the stop codon
• Ribosome dissociates and the polypeptide is released because of changes caused by the release factor in the MRNA TRNA ribosome complex
• Polypeptide folds into functional protein.

E. coli and Gene Regulation

• E. coli, a bacterium residing in colons, possesses approximately 4,000 genes, a subset of which encode diverse proteins.
• The E. coli genome comprises about four million base pairs (A, T, C, and G), prompting the question of how gene expression is regulated.
• An operon is a cluster of genes transcribed as a single RNA molecule, but in AP Biology, it's defined as a mainly prokaryotic gene regulation system with control elements.

Operon Structure

• Operons consist of structural genes (coding for protein), an operator (where a repressor protein binds for regulation), a promoter (where RNA polymerase binds), and a regulatory gene (producing a regulatory protein, often a repressor).

The Tryptophan (Trp) Operon

• The trp operon encodes enzymes for synthesizing tryptophan, an amino acid, and regulates their production.
• In the absence of environmental tryptophan, the always-on regulatory gene produces a regulatory protein unable to bind to the operator, allowing RNA polymerase to transcribe structural genes.
• When tryptophan is present, it binds to the repressor protein, causing a shape change that enables the repressor to bind to the operator, blocking RNA polymerase and halting tryptophan synthesis.
• The trp operon is repressible, with tryptophan acting as a co-repressor.
• This is a negative feedback system because the presence of tryptophan turns off the production of more tryptophan.

The Lactose (Lac) Operon

• The lac operon is an inducible operon encoding enzymes for digesting lactose into glucose and galactose.
• In the presence of lactose, lactose enters E. coli and binds to the repressor protein, changing its shape so it cannot bind to the operator.
• This allows RNA polymerase to transcribe structural genes, producing enzymes that break down lactose and increase cell membrane permeability to lactose.
• In the absence of lactose, the repressor protein binds to the operator, preventing RNA polymerase from transcribing structural genes.
• Lactose is the inducer of the lac operon.
• This is a negative feedback system as lactose induces the system to turn on the production of enzymes to digest it, thereby removing lactose from the system.

Glucose vs. Lactose Metabolism

• E. coli preferentially metabolizes glucose because it's a monosaccharide readily usable in glycolysis.
• When fed both glucose and lactose, E. coli first consumes glucose, resulting in rapid growth.
• After glucose depletion, there's a lag in growth as the lac operon activates to produce lactose-digesting enzymes.

Gene Regulation in Eukaryotes

• In multicellular eukaryotes, gene regulation is complex due to trillions of cells organized into specialized tissues, each with the same DNA but expressing different genes influenced by environmental factors.
• Most eukaryotic DNA is noncoding.
• In eukaryotic cells, most DNA is not expressed being tightly packaged around histones, and methylated, preventing transcription.
• Acetylation loosens DNA, allowing RNA polymerase to transcribe genes.

Epigenetics

• Epigenetics involves reversible chemical modifications of DNA or DNA packaging that alter gene expression without changing the nucleotide sequence.
• Includes methylation of DNA and modifications of histones.
• Epigenetics accounts for the differentiation of tissues during development.
• Epigenetic changes can sometimes be transmitted across generations (intergenerational transmission).
• All cells in an organism are genomically equivalent but differentiate due to expressing different genes, influenced by epigenetic modifications.

Transcription Regulation in Eukaryotes

• Eukaryotes control transcription using regulatory DNA sequences (promoters, enhancers) that interact with regulatory proteins.
• Enhancers increase the probability of gene transcription.
• Interactions between activator proteins, DNA bending proteins, mediator proteins, and general transcription factors enable RNA polymerase to bind, making transcription possible.

Coordination of Gene Expression

• Different tissues express different genes but can share common regulatory sequences, coordinating gene transcription.
• For example, testosterone can induce changes in different tissues via a common testosterone receptor.

Introns and Exons

• Introns are intervening, non-coding DNA sequences within genes, transcribed into pre-mRNA and then removed.
• Exons are DNA sequences that become RNA, ultimately expressed as protein.
• Pre-mRNA needs to be processed, which includes the removal of introns.

Post-transcriptional Modification

• In eukaryotes, pre-mRNA is modified before translation.
• A GTP cap is added at the 5' end, and a poly-A tail is added at the 3' end.
• Introns are excised, and exons are spliced together.
• The 5' GTP cap protects mRNA from breakdown and assists in binding to a ribosome.
• The 3' poly-A tail stabilizes mRNA.

Alternative Splicing

• Through alternative splicing exons can be spliced together in alternate ways.
• Through alternative splicing, multiple protein versions can be produced from the same pre-mRNA transcript, increasing phenotypic variation.
• Exons often code for functional domains within proteins.

Small RNAs in Gene Regulation

• Small RNAs, such as microRNAs, regulate gene expression post-transcriptionally.
• MicroRNAs connect with a protein that's called an RNA silencing complex protein which either degrades mRNA or pauses translation.

Mutations

• A mutation is a random change in DNA or an entire chromosome.
• A point mutation is a change in a single nucleotide.
• Silent mutations are mutations that result in the same amino acid being coded for.
• A nonsense mutation is a mutation that inserts a stop codon instead of an amino acid.
• A missense mutation changes the amino acid from one to another.

Types of Mutations

• Silent mutations: DNA changes, but the same amino acid is coded for (due to genetic code redundancy).
• Nonsense mutations: insertion of a stop codon, prematurely halting protein production.
• Missense mutations: changes one amino acid to another; the effect depends on the chemistry of the substitution.
• Frame-shift mutations: insertions or deletions that alter the reading frame, causing extensive missense or nonsense.

Sickle Cell Disease

• Sickle cell disease is caused by a missense mutation substituting valine (nonpolar) for glutamic acid.
• This substitution causes hemoglobin molecules to stick together under low oxygen conditions, leading to sickled red blood cells and tissue damage.
• The mutation is recessive, but heterozygotes have the sickle cell trait and resistance to malaria.

Effects of Mutations

• Mutations can be positive, negative, or neutral, depending on the environment (contextual).
• Positive mutations increase evolutionary fitness by improving survival and reproduction.
• Negative mutations reduce fitness.
• Neutral mutations have no effect on the phenotype.

Importance of Mutations

• Mutations provide the raw material upon which natural selection acts.
• Mutation makes evolution a creative process that results in adaptation.

Germline vs. Somatic Mutations

• Germline mutations occur in cells that make gametes and are present in every cell of the offspring, and can be inherited.
• Somatic mutations emerge in tissues during development or adulthood, and only affect the organism and are not passed on.

Horizontal Gene Transfer

• Horizontal gene transfer involves one organism transferring genes to another organism that is not its offspring.
• Unlike vertical gene transfer, where parents transmit genes to offspring.
• In unicellular organisms, newly acquired genes become part of the recipient's genome.

Bacterial Conjugation

• Bacteria transfer genes via conjugation which occurs when the pilus contacts a second cell.
• Plasmids with genes for a pilus are copied/transferred to another bacterium.

Bacterial Transformation

• Bacteria pick up DNA fragments from the environment.

Viral Transduction

• Viral transduction involves viruses transferring genes between organisms.
• During viral infections, the virus breaks the hosts genome and breaks apart the host's genome.
• New viral particles can carry DNA from the original host.

Horizontal Gene Transfer and Viral Recombination

• Horizontal gene transfer can occur through viral transduction in which mistakes in the viral replication cycle result in host DNA being incorporated into a virus and transferred to another cell. Viral recombination, where different viral strains infect the same host, can lead to new viral strains and potential pandemics.

Recombinant DNA

• Recombinant DNA is DNA combined from multiple sources.
• Restriction enzymes recognize specific DNA sequences.
• Restriction enzymes creates restriction fragments by cutting DNA at specific sequences, leaving sticky ends for base pairing facilitating the creation of recombinant DNA with DNA ligase.

Creating Recombinant DNA

• A plasmid from a bacterial cell and cutting open that plasmid with restriction enzyme leaving sticky ends
• The same restriction enzyme is used to cut out a Target human gene and therefore the ends will be complimentary will be able to bond together.
• Recombinant plasmid needs to be inserted into an bacterial cell using transformation.

Need for Intron Removal for Expression

• Genes for human proteins: Before genes from human proteins such as insulin can be expressed in bacteria, introns need to be removed to allow translation.
• Introns can be removed either by biochemically reverse engineering DNA or using reverse transcriptase.

Gel Electrophoresis

• Gel electrophoresis sorts molecules by size and charge.
• Restriction fragment analysis/DNA fingerprinting can be used for forensics.

Polymerase Chain Reaction

• In PCR, polymerase Chain Reaction, a cell-free technique is used for cloning DNA with DNA polymerase, primers, heat resistant DNA polymerase, and free nucleotides.
• The repeated cycles of Heating and Cooling will double the amount of DNA.

DNA Sequencing

• DNA sequencing involves figuring out the specific sequence of nucleotides of a sample DNA from a small fragment to the entire Genome of an organism.
• DNA sequencing allows biologists to determine what proteins an organism can produce and it's used to infer evolutionary relationships.