AP Biology: DNA, RNA, and Gene Expression

• This video prepares students for the AP Bio exam or a unit 6 test.
• Complex topics like RNA production, modification into mRNA, and translation into protein are simplified.
• DNA and RNA structure and function are covered.
• DNA replication, transcription, and translation are covered.
• The video explains the genetic code.
• Gene expression regulation in prokaryotes focusing on operons is explained.
• Eukaryotic gene expression is detailed.
• Mutation and horizontal gene transfer are explained.
• The video includes information about biotechnology.
• Glenn Wolkenfeld, also known as Mr. W, created the video.
• Mr. W is a retired AP biology teacher.
• A downloadable checklist is available at AP bios to help study.

DNA Structure

• DNA is a double-stranded helical molecule composed of nucleotide monomers.
• Each strand contains deoxyribose sugars and phosphate groups, forming the sugar-phosphate backbone.
• Nitrogenous bases within the helix bind through hydrogen bonds, following specific base pairing rules.
• Adenine (A) binds only with thymine (T), while guanine (G) binds only with cytosine (C).
• The two DNA strands are anti-parallel, with one strand oriented upside down relative to the other.

DNA as Hereditary Molecule

• The sequence of bases in DNA serves as an informational code, specifying RNA and protein sequences.
• Base pairing (A-T, G-C) allows each strand to serve as a template for synthesizing a complementary strand during replication.
• DNA's double helical structure protects the bases inside, enhancing stability.
• DNA is capable of mutation, with bases changing spontaneously or due to environmental factors, enabling evolution.

Comparison of DNA and RNA Functions

• DNA serves as the hereditary molecule in all cell-based life forms.
• RNA is the hereditary molecule in some viruses (e.g., HIV, SARS-CoV-2).
• RNA is involved in information transfer related to protein synthesis.
• Types of RNA include mRNA, tRNA, and rRNA.
• In eukaryotes, RNA regulates gene expression, including splicing out introns from pre-mRNA to create mRNA.

Genetic Information Storage in Prokaryotes and Eukaryotes

• Prokaryotes store DNA in looped circular chromosomes without protein scaffolds.
• Prokaryotic genomes range from 100,000 to 10 million base pairs.
• Eukaryotes organize DNA into multiple linear chromosomes wrapped around histones.
• Eukaryotic genomes are larger than prokaryotic genomes; for example, the human genome contains 3.2 billion base pairs.

Plasmids

• Plasmids are small, extra-chromosomal loops of DNA commonly found in bacteria.
• Plasmids are involved in horizontal gene transfer between bacterial cells through conjugation, transferring genes, including those for antibiotic resistance.
• Plasmids are used in genetic engineering for replicating DNA and expressing engineered genes within bacterial cells.

DNA Replication Overview

• During DNA replication, enzymes use each strand of the double helix as a template to synthesize new daughter strands.
• Each daughter DNA double helix consists of one conserved strand from the parent molecule and one newly synthesized strand.
• Semiconservative replication is the name for this process.

Initiation of DNA Replication

• The process begins when helicase finds the origin of replication and separates the double-stranded DNA.
• This creates a replication fork, exposing two single strands.

Key Enzymes: DNA Polymerase, Primase, and Primers

• DNA polymerase is the key enzyme for creating new DNA strands.
• DNA polymerase binds new nucleotides to the 3' end of a growing strand.
• DNA polymerase requires an RNA primer to begin adding nucleotides.
• Primase lays down this initial RNA primer.

Single-Strand Binding Proteins

• Single-strand binding proteins keep the double helix from rewinding during replication.

Leading vs. Lagging Strand

• In the leading strand, DNA replication is relatively continuous because DNA polymerase follows the opening replication fork.
• In the lagging strand, DNA polymerase synthesizes in the opposite direction, resulting in discontinuous replication and Okazaki fragments.

DNA Polymerase 1 and Ligase

• DNA polymerase 1 removes RNA primers and replaces them with DNA.
• DNA ligase seals the gaps between fragments with sugar-phosphate bonds.

Central Dogma of Molecular Genetics

• Explains the overall flow of genetic information within cells.
• DNA makes RNA makes Protein
• Information flows from a sequence of DNA triplets to a sequence of mRNA codons to a sequence of amino acids.

Definition of a Gene

• A gene is the basic unit of heredity passed from parent to offspring.
• It determines a trait in terms of molecular genetics.
• It's a sequence of DNA nucleotides that codes for RNA, which codes for protein.

Principle Forms of RNA

• mRNA (messenger RNA) brings instructions from DNA to ribosomes.
• rRNA (ribosomal RNA) makes up the catalytic part of ribosomes and binds amino acids together during protein synthesis.
• tRNA (transfer RNA) brings specific amino acids to the ribosomes for protein synthesis.
• small RNAs are involved in eukaryotic gene regulation.

Transcription

• Transcription is the creation of RNA from DNA.
• Every gene begins with a promoter region that indicates where the gene starts.
• During transcription, RNA polymerase binds with the Promoter on DNA.
• RNA polymerase transcribes the sequence of DNA bases on DNA's template strand into a sequence of RNA.
• RNA polymerase reads the DNA in the 3' to 5' direction and synthesizes new RNA in the 5' to 3' direction.
• When the RNA polymerase reaches a Terminator region it dissociates from the DNA, ending transcription.

Template Strand

• The template strand is the strand gets transcribed from DNA into RNA.
• The template strand is also called The noncoding Strand, The antient Strand and the minus strand.
• The complementary strand to the template strand is called the coding strand, because it has the same sequence of nucleotides as the mRNA.
• The coding strand is called the sense strand or the positive strand.

Unique Features of Prokaryotic Transcription

• Procaryotes don't have a nucleus, so there's no separation between the genetic material and the cytoplasm.
• Transcribed RNA can immediately be translated by ribosomes into protein.
• Multiple ribosomes often read the same RNA strand, which are called polysomes.

Genetic Code

• Genetic code is the code used by to translate nucleotide sequences into amino acid sequences.
• DNA to RNA is transcription and RNA to protein is the genetic code.
• Groups of three RNA nucleotides are called codon, and they code for one amino acid.
• The code is nearly universal, specific and redundant.

Translation

• mRNA contains the codons specify the order of amino acids.
• The ribosome connects amino acids to create a polypeptide.
• tRNAs brings amino acids to this ribosome mRNA complex.
• tRNAs have an anti-codon.

Role of the Ribosome

• Ribosomes are general-purpose protein factories.
• Ribosomes string together amino acids to form polypeptides.
• Follows the instructions in mRNA.
• Ribosomes have a large and a small subunit.
• Ribosomes have three tRNA-binding sites: the E, P, and A site.

Translation pt 2

• Processed mRNA, leaves the nucleus and binds with the small ribosomal subunit
• Small ribosomal subunit makes its way to the start codon, which is AUG that's where translation begins
• Small subunit the waits for a tRNA with a matching anti-codon to bind with the Starcon
• The first TRNA is carrying the first amino acid methionine, then the large subunit binds with the small subunit
• The ribosome is now complete and that first TRNA with methionine is located in the ribosomes P site

Elongation Phase of Translation

• The next TRNA comes to the a-site and it Bears a new amino acid
• The ribosome then catalyzes a peptide bond between the p and a site amino acids
• The ribosome then translocates and moves over one more codon, so that a dipeptide is now hanging off the psite amino acid
• After the ribosome moves over one more codon, then the a side is empty and there's a TRNA in the E site which is not connected to the polypeptide
• TRNA that's in the ester exits and the new TRNA enters at the a-site
• A new TRNA comes in, then the ribosome catalyzes a bond between that new amino acid and the existing dipeptide

Termination Phase of Translation

• The ribosome gets to a stop codon and those stop codons and the genetic code have no corresponding TRNA
• Instead what they do is they code for a release Factor That's A protein that can bind with the stop codon
• Changes in the MRNA TRNA ribosome complex and those changes cause the ribosome to dissociate and that polypeptide to be released
• Fold polypeptide into a functional protein and translation is done

Operons

• An operon is a cluster of genes transcribed as a single RNA, mostly found in prokaryotes.
• It's a system of gene regulation with control elements allowing for gene regulation.
• The structure includes structural genes (code for proteins), an operator (where a repressor binds), a promoter (where RNA polymerase binds), and a regulatory gene that produces a regulatory protein (typically a repressor).

The Tryptophan (trp) Operon

• Codes for enzymes that synthesize tryptophan.
• It is a regulatory system that turns on enzyme production only when tryptophan is scarce.
• If there's no tryptophan, RNA polymerase binds to the promoter and transcribes structural genes.
• Tryptophan in the environment binds to the repressor protein, changing its shape.
• This allows the repressor to bind to the operator, blocking RNA polymerase.
• Operon only synthesizes Tryptophan when Tryptophan levels are low
• The trp operon is repressible, and tryptophan acts as a corepressor.
• It is a negative feedback system, turning off when tryptophan is abundant.

The Lactose (lac) Operon

• Codes for enzymes that digest lactose (a disaccharide).
• The enzymes break lactose into glucose and galactose.
• Lactose diffuses into e.coli and binds to the repressor protein when present.
• This changes the shape of the repressor, preventing it from binding to the operator.
• RNA polymerase can then transcribe the structural genes.
• When lactose is absent, the repressor binds to the operator, blocking transcription.
• The lac operon is an inducible system, activated by lactose (the inducer).
• It is a negative feedback system; when lactose is digested, the system turns off.

E.coli Metabolism of Glucose and Lactose

• E.coli prefers to metabolize glucose because it's a monosaccharide.
• The bacteria digests glucose first, growing rapidly in the process.
• Once glucose is depleted, there's a lag during the activation of the lac operon.
• After the lac operon is activated lactose-digesting enzymes are produced to break down lactose.
• A similar lag will occur once lactose is depleted.

Eukaryotic Gene Regulation

• Involves multicellular organisms with trillions of cells in specialized tissues.
• Each cell has the same DNA, but gene expression varies based on the cell's function.
• Cells need to know which genes to express during development.
• This is determined by the cell type and environmental factors.
• Most eukaryotic DNA is noncoding, with genes containing introns.

Epigenetics and Gene Expression

• In any given cell, most DNA is silenced and not expressed.
• Tightly packaged DNA around histones and methylation prevent transcription.
• Acetylation loosens DNA, allowing RNA polymerase to transcribe genes.
• Epigenetics are reversible chemical modifications of DNA or its packaging.
• Epigenetics doesn't change the DNA sequence, but affects its expression.
• It's responsible for cell differentiation and tissue specialization.
• Epigenetic changes can sometimes be transmitted across generations.

Gene Expression Coordination

• Different tissues express different genes but can share regulatory sequences.
• Common regulatory sequences coordinate gene transcription in various tissues.
• An example is the testosterone receptor gene shared between tissues in male lions.
• Testosterone binds to the receptor, activating different genes in different tissues.

Introns, Exons, and mRNA Processing

• Introns are intervening sequences within genes, transcribed into pre-mRNA but removed before translation.
• Exons are DNA sequences that become RNA, ultimately expressed as protein.
• Pre-mRNA undergoes post-transcriptional modifications in eukaryotes.
• These include the addition of a 5' GTP cap and a 3' poly-A tail.
• Introns are excised, and exons are spliced together to form mRNA.

Function of 5' Cap and 3' Poly-A Tail

• The 5' GTP cap protects mRNA from breakdown and aids in ribosome binding.
• The 3' poly-A tail stabilizes mRNA and delays enzymatic breakdown.

Alternative Splicing and Phenotypic Variation

• Through alternative splicing, exons are spliced together in alternative ways.
• This produces multiple protein versions from the same pre-mRNA transcript.
• These exons are able to code for functional domains, that when put together make slightlty different functions
• Each exon codes for functional domains, creating proteins with slightly different functions and increasing phenotypic variation.

Small RNAs and Eukaryotic Gene Regulation

• Small RNAs, such as microRNAs (miRNAs), regulate gene expression.
• MicroRNAs play a role in post-transcriptional control of gene expression through RNA silencing.
• DNA contains genes that code for microRNAs.
• Pre-microRNA needs to be processed to a mature micro RNA.
• RNA silencing involves microRNAs interacting with RNA silencing complex proteins.

Mechanisms of RNA Silencing

• If the microRNA completely matches an mRNA sequence, it causes degradation and destruction of the mRNA.
• If there's a partial match, the complex pauses or blocks translation.
• The system has effectively changed gene expression through micro RNA.

Mutations: An Overview

• A mutation is a random change in DNA or an entire chromosome.
• A point mutation is a change in a single nucleotide.

Types of Point Mutations

• Silent mutations result in the same amino acid being coded for, due to genetic code redundancy.
• Nonsense mutations insert a stop codon, halting protein production.
• Missense mutations change the amino acid coded for.

Consequences of Missense Mutations

• The effect depends on the chemistry of the substituted amino acid.
• A conservative missense mutation may have minimal impact if the chemistry is similar.
• A more significant change occurs with substitutions that drastically alter chemistry.

Frameshift Mutations

• Result from insertion or deletion of nucleotides, altering the reading frame.
• These can lead to extensive missense or nonsense, disrupting protein production.

Sickle Cell Disease

• Caused by a single substitution mutation.
• Valine (nonpolar) replaces glutamic acid, which has a big significance on the chemistry of hemoglobin.
• This affects the hemoglobin protein, causing it to stick together under low oxygen conditions.
• This leads to sickled or spiked red blood cells that cause tissue damage.

Effects of Mutations

• A mutation's effect depends on the environment.
• A positive mutation improves a phenotype, increasing evolutionary fitness.
• A negative mutation reduces evolutionary fitness.
• In the context of malaria ridden area, having one does of sickle cell aliel leads to resistance of malaria.
• A neutral mutation has no effect on the phenotype, occurring in non-coding regions or resulting in silent mutations.

Importance of Mutations to Evolution

• Mutations provide the raw material upon which natural selection acts.
• Without mutation, natural selection can only cull harmful variants.
• With mutation, new variants arise that can increase a population's fitness.

Germline vs. Somatic Mutations

• Germline mutations occur in cells that produce gametes and so are present in all cells of the offspring.
• Somatic mutations emerge in some tissue and only affect the organism; they are not passed down.

Horizontal Gene Transfer

• Horizontal gene transfer is when one organism transfers genes to another organism that is not its offspring.
• Vertical gene transfer is when the parents transmit their genomes to their offspring.

Bacterial Conjugation

• Bacteria transfer genes via a plasmid loop of DNA through a membrane extension.
• The pilus contracts a second cell, copying and transmitting the plasmid to that recipient.
• Conjugation spreads antibiotic resistance genes through bacterial populations.

Bacterial Transformation

• Bacteria pick up DNA fragments from the environment, incorporating them into their genome.
• This DNA can include plasmids.
• Transformation is useful in genetic engineering to introduce foreign genes into bacterial cells.

Horizontal Gene Transfer Through Viral Transduction

• Virus breaks host's genome, the virus then uses the cell's molecular machinery to create new viral genes and proteins.
• DNA fragments are mistakenly incorporated into a virus.
• That virus infects a cell in another organism and brings in foreign DNA.
• DNA recombines with the DNA in the new host, incorporating new genes.

Viral Recombination

• Two different strains of viruses infect the same host.
• The genes of the viruses can recombine.
• Emergence of new viral strains can occur.
• Animal immune systems can't recognize, leading to pandemic viral outbreaks.

Recombinant DNA

• DNA from more than one source artificially combined in a laboratory.
• Can have bacterial DNA + human DNA + bacterial DNA
• Restriction enzymes, which find sequences of DNA called restriction sites.
• Restriction enzymes cut DNA at restriction sites, producing restriction fragments with sticky ends.
• Complimentary sticky ends pair via hydrogen bonds.
• DNA ligase creates sugar-phosphate bonds, connecting strands and creating recombinant DNA.

Creating Recombinant Plasmids with a Human Gene

• Extract a plasmid from a bacterial cell and cut it open with a restriction enzyme.
• Use the same enzyme to cut out a target human gene, creating complimentary ends.
• The human gene combines with the plasmid, forming hydrogen bonds.
• DNA ligase binds the human and plasmid DNA, creating a recombinant plasmid with a human gene.
• Insert the plasmid into a bacterial cell via transformation.
• The genetically engineered bacteria produces a human protein every reproduction cycle.

Introns and Expression of Human Proteins in Bacteria

• Introns are non-coding sequences within eukaryotic genes that have to be spliced out before the genes RNA can be translated.
• You have to use DNA from which the introns have been removed so that the bacterial protein can be translated
• Linear sequence of amino acids is reverse engineered using the chart to then get to the DNA.
• Extract mRNA from those cells that codes for this protein with reverse transcriptase that's in HIV.
• Reverse transcriptase uses cdna and rna dna compliment and then is inserted into the bacterial cells plasmid.

Gel Electrophoresis

• Sorts molecules by size and/or electrical charge.
• DNA's phosphate groups are negatively charged, DNA fragments will move away from the negatively charged side of the field.
• Smaller fragments are impeded less than larger fragments, enabling separation by size.
• Used for restriction fragment analysis, or DNA fingerprinting.

Restriction Mapping Example

• When digest the plasmid, restriction enzymes create specific DNA fragments.
• Restriction sites need to analyzed and the plasmid by calculating sizes
• Corresponding fragments need to be selected by calculating the known distance between the restriction sites

Polymerase Chain Reaction (PCR)

• Cel-free technique for cloning DNA.
• Needs DNA sample, primers short single stranded sequences of DNA.
• Heat resistant, so the DNA Polymerase won't be denatured.
• Needs free nucleotides that are going to be used for DNA synthesis
• DNA polymerase will read the template Strand and it'll seal sugar phosphate bonds between the nucleotides