Chapter 17-21

17.1 Genes Specify Proteins via Transcription and Translation

• Gene expression: process by which DNA directs synthesis of proteins (rarely, RNA).

• Not all proteins are enzymes, but all enzymes are proteins.

• DNA: AT CG; RNA: AU CG;

• Each polypeptide of protein has monomers (amino acids) in a particular linear order (primary structure).

• Nucleic acids and proteins contain information, except in different languages.

Basic Principles of Transcription and Translation

• Transcription: Synthesis of RNA using information in the DNA. DNA information is “transcribed” into RNA language.

  • Messenger RNA (mRNA): has a sequence that is complementary to the DNA template and carries the genetic instructions from the nucleus to the ribosomes for protein synthesis.

  • Pre-mRNA: The initial RNA transcript that contains both introns and exons, which undergoes splicing to remove introns and join exons before becoming mature mRNA.

• Translation: Synthesis of a ammino acid polypeptide using information from the mRNA which traveled to a ribosome in the cytoplasm.

• Bacteria do NOT have a defined nucleus, thus their transcription and translation occur simultaneously in the cytoplasm.

Genetic Code

• Codons or Triplets of Nucleotides: Each codon consists of three nucleotides that specify a particular amino acid or signal the termination of protein synthesis.

• Template strand: The DNA strand that is complementary to the coding strand and serves as a guide for RNA polymerase during transcription.

• mRNA is COMPLEMENTARY not identical to it’s DNA template due to base-pairing rules.

• Coding strand: The DNA strand that has the same sequence as the mRNA transcript, except that thymine is replaced by uracil in RNA.

• Sixty-one out of sixty-four codons code for ammino acids, while the there are three stop codons that signal the termination of protein synthesis.

  • All codons must have a Methyl (AUG) beginning.

17.2 Closer look at Transcription

• RNA polymerase: pries two DNA strands apart from each other and joins the RNA nucleotides complementary to DNA template strand together.

  • Interesting that bacteria only have one RNA polymerase which accounts for all enzyme functions while eukaryotes have at least three types of RNA polymerase in the nuclei.

• Promoter: a specific DNA sequence that signals the beginning of transcription and where RNA polymerase binds to initiate the process.

  • In Bacteria, there is a terminator, which signals the end of transcription.

• Transcription Unit: Stretch of DNA downstream from promoter that gets transcribed into an RNA molecule.

• RNA polymerase II: Synthesizes pre-mRNA.

RNA Polymerase the nitty and gritty

• Start point: Within the promoter, this is the actual start point of the synthesis of pre-mRNA.

• Transcription factors (in eukaryotes): Collection of proteins which help guide the binding of RNA polymerase and initiation of transcription.

• Transcription initiation complex: After transcription factors are bound, RNA polymerase II will bind to the promoter area.

• TATA box: crucial promoter region that provides a binding site for transcription factors and RNA polymerase II, ensuring proper initiation of transcription.

Elongation!

• RNA polymerase moves along the DNA, untwisting the double helix, exposing 10-20 DNA nucleotides for pairing with RNA nucleotides.

• Single gene can be transcribed simultaneously by several molecules of RNA polymerase.

Termination of Transcription!

• Within bacteria, transcription proceeds through a terminator sequence in the DNA.. making polymerase detach and release the transcript.

• Within eukaryotes, RNA polymerase II transcribes sequence on DNA polyadenylation signal sequence, which signals for immediate attaching of specific proteins shortly after that cut pre-mRNA free from polymerase.

  • RNA polymerase II continues transcribing until it dissociates and enzymes eat up the waste.

Concept 17.3 Eukaryotic Cells modify RNA after transcription

• RNA processing: Both ends of primary transcript altered, and interior sections are cut out and put together.

• 5’ end which is synthesized first, receives a 5’ cap that consists of a modified guanine nucleotide, helping to stabilize the RNA and facilitate its recognition by ribosomes during translation.

  • 5’ UTR is a region between 5’cap and start codon that will not be translated, but helps with ribosome binding.

• The 3’ end is also modified by the addition of a poly-A tail, which enhances the stability of the mRNA and aids in the export of the transcript from the nucleus to the cytoplasm.

  • 3’ UTR: This makes up the polyadenylation signal that the poly-A tail binded to.

• The 5’ modified guanine nucleotide cap and 3’ end poly-adenine nucleotide tail serve to facilitate export of mature mRNA from nucleus, protect mRNA from degradation by hydrolytic enzymes and help ribosomes attach to 5’ end of mRNA.

Split Genes and RNA Splicing

• RNA splicing: large portions of the RNA primary transcript molecules are removed and remaining portions reconnected.

  • Introns: noncoding segments of nucleic acid which lie between coding regions.

  • Exons: expressed sequences of RNA that are included in the final mature RNA molecule after splicing.

• Spliceosome: Large complex of proteins which binds to several short nucleotide sequences along an intron, and key ones. Introns are thrown out and degraded rapidly and two exons are put together.

• Ribozymes: RNA molecules which function as enzymes.

Functional Importance of Introns

• Alternative RNA splicing: Genes can give rise to two or more different polypeptides, depending on which segments end up as exons during processing.

• Domains: Proteins have modular architecture which allows for structural and functional regions.

• Exon shuffling: this process can lead to the creation of new proteins with distinct functions by mixing and matching the exons from different genes.

  • Introns increase this by allowing more terrain, allowing for easier crossin.

Concept 17.4 Translation is RNA-directed synthesis of a polypeptide.

• tRNA: transfer an amino acid from cytoplasmic pool of amino acids to a growing polypeptide in a ribosome.

  • A single long RNA molecule that has a anticodon which corresponds to a specific amino acid on a mRNA strand.

  • The 3’rd end of the tRNA has a amino acid attached to it.

• Cells keep all 20 amino acids stocked in their cells.

Ribosomes

• Ribosomes contain a lower subunit which binds the mRNA strand and feeds it into the ribosome, while the upper subunit facilitates the formation of peptide bonds between the amino acids.

• The upper subunit contains three binding sites for tRNA.

  • A (aminoactyl-RNA binding) site binds tRNA holding the next amino acid to be added to the chain.

  • P (peptidyl-tRNA binding) site: Holds tRNA carrying the growing polypeptide chain.

  • E (exit) site: Discharged tRNA, which is no longer attached to an amino acid, exits the ribosome after its role in protein synthesis is fulfilled.

Polypeptide Production in Steps

1. mRNA binds to mRNA binding site on smaller subunit.

2. Initiator tRNA with anticodon UAC, binds with the MET on mRNA.

3. tRNAs attempt to bind to the A site, where if base-pairing happens, it attaches.

4. Large subunit then transfers the amino acid peptide to the tRNA in A site.

5. The uncharged tRNA in P site is moved to E site and the charged tRNA in A site is moved to P site.

Termination.

• Once the ribosome hits a stop codon on mRNA, A site changes to allow a “release factor” which binds to the stop codon.

• Binding to stop codon, promotes hydrolysis of bond between tRNA in P site and amino acid peptide change.

• Then the ribosome dissociates.

Free and Bound Ribosomes and Polypeptide locations

• Free ribosomes: Synthesize proteins that function within the cytosol

• Bound ribosomes: Synthesize proteins destined for secretion or membrane insertion.

• SRP or Signal-recognition particle HALTS synthesis of a polypeptide chain in a free ribosome, then drags the ribosome and all to the rough endoplasmic reticulum (RER) where protein synthesis resumes.

• Peptide gets through into the ER Lumen afterwards.

Production of Multiple Polypeptides in Bacteria and Eukaryotes.

• Single mRNA can be used to make many copies of a polypeptide simultaneously.

• So a single mRNA can feed through 4 ribosomes at once.

• These are called POLYRIBOSOMES or Polysomes and can be free or bound.

• Bacteria can simultaneously translate mRNA, and transcribe it multiple times over. Allowing for a single gene to produce numerous protein copies in a highly efficient manner.

Concept 17.5 MUTATIONS!!!

• Mutation are changes in genetic information of a cell.

• Point mutations which are small-scale changes can result in the alteration of a single nucleotide base in the DNA sequence.

  • Sickle cell.

Small Scale-Mutations

• Substitutions: Nucleotide-pair substitution: replacement of one nucleotide and its partner with another pair of nucleotides.

  • Missense mutations: Change one amino acid into another amino acid.

  • Silent mutation: Simple. No effect on phenotype.

  • Nonsense: A mutation that results in a premature stop codon, leading to truncated and usually nonfunctional proteins.

• Insertions and Deletions: Nucleotide pairs are either added or deleted.

  • Disastrous effect typically.

  • Frameshift mutation: Caused when number of nucleotides inserted or deleted is not a multiple of three. This causes improper grouping of codons downstream.

  • 3-nucleotide-pair deletion: This type of deletion removes a complete codon, which can lead to the loss of a single amino acid in the resulting protein, potentially altering its function.

Chapter 18

Operons:

• Operator: On and off switch of an gene. Controls access of RNA polymerase to genes.

• Repressor: Binds to the operator, preventing RNA polymerase from transcribing genes.

  • Regulatory gene: Codes for a repressor protein that can bind to the operator and control the expression of the operon and typically has it’s own promoter too.

Forms of Negative Feedback Regulation

• Inducer: A molecule that binds to the repressor, causing it to change shape and release from the operator, thereby allowing RNA polymerase to transcribe the genes.

• Copressor: A molecule that assists a repressor in binding to the operator, further inhibiting gene transcription when present.

Positive Gene Regulation

• Activator: Protein which binds to DNA and stimulates transcript of a gene.

  • cAMP is an example of a signaling molecule that activates the CAP (catabolite activator protein), which in turn enhances the binding of RNA polymerase to the promoter, facilitating gene transcription quicker.

  • Lower affinity by default, however an activator increases affinity, increasing it’s rates.

Concept 18.2 Eukaryotic gene expression is regulated at many stages

• Differential gene expression: Expression of different genes by cells with the same genome.

• Transcription factors MUST find the right genes to express at the right time, otherwise.. cancer and imbalances can occur.

• Heterochromatin: Much more densely arranged chromatin.

• euchromatin: A less densely packed form of chromatin that is associated with active gene transcription and is more accessible to transcription factors.

Histone Modifications and DNA Methylation

• N-terminus: The end of a protein or polypeptide that is characterized by a free amino group, playing a crucial role in the folding and function of the protein.

• Histone tails or N-terminus of a histone allows for modifying enzymes that catalyze addition or removal of specific chemical groups like acetyl, methyl, and phosphate groups.

  • Histone acetylation: Addition of acetyl group to amino acid in histone tail promotes transcription by opening up of chromatin structure.

  • Histone Methylation: Addition of a Methyl group to amino acid in histone tail promotes condensation of chromatin and reduced transcription.

• Nucleosomes are like gum balls with spider legs, and those spider legs are histone tails. The DNA double helix goes around them and binds them.

Epigenetic Inheritance

• Epigenetic Inheritance of traits transmitted by mechanisms not involving the nucleotide sequence itself.

• Mutations in DNA are permanent, however modifications to chromatin can be reversed.

• These mutations can lead to changes in gene expression that may affect phenotype, sometimes even across generations.

Regulation of Transcription Initiation

• Chromatin-modifying enzymes provide initial control of gene expression by making a region of DNA more or less able to bind transcription machinery.

Eukaryotic Gene and Transcript Organization

• Control elements: segments of noncoding DNA that serve as binding sites for proteins called transcription factors, which help regulate the transcription of nearby genes.

• General Transcription Factors at the Promoter are the essential for transcription of all protein-coding genes.

  • Protein-Protein interactions important here. Only when complete initiation complex is formed, can polymerase actually move along the DNA template strand and transcribe.

Enhancers and Specific Transcription Factors

• Proximal and distal control elements are simply factors near promoter.

• Specific Transcription Factors: proteins that bind to specific DNA sequences to enhance or inhibit the transcription of particular genes, allowing for precise regulation in response to cellular signals.

• Enhancers: usually made up of distal control elements that can significantly increase the likelihood of transcription when bound by specific transcription factors.

• Basically Special Transcription Factors bind to Enhancers… enhancers are not essential.

Post-transcriptional Regulation

• Alternative RNA splicing: different mRNA molecules are produced from same primary transcript, depending on what segments get treated as exons and introns.

• Regulatory proteins specific to a cell-type control the intron/exon choices through binding to regulatory sequences.

Differentiation 18.4

• Cell differentiation → Cell changes structure and form into a mature different cell.

• Morphogenesis → Processes which give an organism its shape.

• Cytoplasmic determinants → Maternal substances inside the egg which influence course of early development.

• Induction → External cells release signaling molecule which encourages cell to change gene expression.

• Determination → The process by which cells become committed to a specific fate, leading them to develop into particular cell types or tissues.

Chapter 20!

• DNA Technology: Techniques for manipulation of DNA.

• Nucleic acid hybridization: Process of base pairing of one strand of a nucleic acid to a complementary sequence from another nucleic acid strand, either DNA or RNA.

• Genetic engineering: Direct manipulation of genes, often using nucleic acid hybdrization.

• DNA sequencing : A method used to determine the exact sequence of nucleotides in a DNA molecule, which is essential for understanding genetic information and variations.

Copying a Gene or DNA segment

• DNA cloning: Specific segments of DNA is analyzed and replicated to produce identical copies.

• Often bacteria is used. Bacteria usually have plasmids, which is small circular DNA molecules that are replicated separately.

  • Scientists will isolate plasmids from bacterial cells and alter them by genetic engineering, allowing them to insert DNA they wish to study.

  • Once a plasmid is altered, it is a recombinant DNA molecule, a molecule containing DNA from two different sources.

  • Plasmids act as a cloning vector, and are used in “vitro” for studies.

• Gene cloning is useful for: to make many copies of, a specific gene for further study, producing proteins of interest, and creating genetically modified organisms (GMOs) that can exhibit desired traits.

Restriction Enzymes to make Recombinant DNA Plasmid

• Restriction enzymes: protect bacterial cells by cutting up foreign DNA from organisms and phages.

• Restriction site: Restriction enzymes have a specific short DNA sequence they recognize.

• DNA is protected from these own restriction enzymes through methyl groups to adenines or cytosines

• Restriction fragments: The resulting fragments left behind. Usually this leaves behind sticky ends that can easily anneal with complementary DNA fragments.

• Gel electrophoresis: Uses a gel made of a polymer that has microscopic holes of different sizes, through which shorter fragments travel faster.

Amplifying DNA: Polymerase Chain Reaction (PCR)

• Polymerase chain reaction: Replicates billions of copies of a specific target DNA segment in a sample.

Cycle 1:

• Denaturation: Heat briefly to separate strands.

• Annealing: Cool to allow primers to form hydrogen bonds with ends of target sequence.

• Extension: DNA polymerase adds nucleotides to 3’ end of each primer.

Cycle 2: yields 4 molecules; Cycle 3 yields 8, 2 of which are identical.

Expression of Cloned Eukaryotic Genes

• Expression vector: a cloning vector that contains highly active bacterial promoter just upstream of a restriction site where a eukaryotic gene can be inserted.

• Eukaryotic cells present issues with introns.

• Yeast will usually be used since it is a single-celled eukaryotic cell with plasmid.

• Electroporation: brief electrical pulse applied to solution containing cells creates temporary holes in their plasma membranes, which DNA can enter.

20.3: fuck!!!!

• Stem cell: relatively unspecialized cell which can reproduce itself indefinitely, and under right conditions differentiate into specialized cells.

• Totipotent: a type of stem cell that has the potential to develop into any cell type in the body, including both placental and embryonic tissues.

• Nuclear transportation / somatic cell nuclear transfer (eukaryotic cells): eggs are enucleated (nucleus removed) and then their nucleus is replaced with a modified one.

  • Eukaryotic cells can use cells from other eukaryotic cells’s nucleus. Can’t be too diff

Cloning Human Stem Cells

• Attempts have been made… not for human replication but for stem cell replication.

• Stem cells in the Human Body can either divide into another stem cell or differentiate.

• Embryonic stem cells can generate all embryonic cell types.. which differentiate into liver, nerve, blood cells and more.

  • Pluripotent.

• Adult stem cells generate only a limited number of cell types.

• Objective is Therapeutic cloning. Issue is intensive costs and ethics.

  • Ethics: 2021 cloned macaque embryos had human cells injected. 3 out of 91 died, and they only contained roughly 5% of human cells. Obj was to grow human organ in monkey.

• Induced Pluripotent Stem (iPS) cells → Modified retrovirus used to deprogram differentiated stem cells back into a pluripotent state. This is legitimate, however some differences are present such as in gene expression.

  • Human iPS cell lines of diseased patients can be used to find the cause of disease at origin AND can be used in the patient itself to promote healthy fixing of cells.

Concept 20.4: Practical applications of DNA-based biotechnology affect our lives

• Biotechnology: Manipulation of organisms or their components to make useful products.

• Medical Application → Comparing RNA-seq and DNA with healthy and diseased cells can help with diagnosing and targeting specific treatments that address the underlying genetic causes of diseases.

  • PCR can be used to replicate and use nucleic acid probes to track down pathogens. RT-PCR can be used to find Covid-19 RNA.

• Personal Genome Analysis → Individuals can be tested by PCR and sequenced for a SNP that is correlated with abnormal genes.

• Human Gene Therapy → Introduction of genes into a afflicted individual for therapeutic purposes. RNA inserted into cloned gene, into a viral capsid, and virus infects bone marrow cells after injection, fixing the gene.

• Transgenic genes → genes converted from one organism into another, allowing for the introduction of desired traits or functions that may enhance resistance to diseases or improve growth and development.

Forensic Evidence and Genetic Profile

• Genetic profile: individual’s unique set of genetic markers.

• Short tandem repeats (STRs): sequences of DNA that are repeated in tandem and are used as important markers in forensic analysis to differentiate between individuals.

Ethics again

• Shockingly lack of concern on recombinant microorganisms but concern on Genetically Modified Organism (GMOs) for food.

• GMO is a transgenic organism that has acquired one or more genes from another species, allowing for specific traits to be expressed.

• Fears that GM crops can give genes to other plants, for instance, “super weeds” could form pollen transfer of genes.

CHAPTER 21!!

Chapter 21.1: Human Genome Project and sequencing techniques.

• Genomics: The study of the complete set of DNA, including all of its genes, which aims to understand the structure, function, evolution, and mapping of genomes.

• Human Genome Project: Began in 1990, its purpose was to determine the sequence of the human genome and identify the genes it contains, thereby providing insights into genetic disorders and human biology.

  • Largely completed by 2003, with each chromosome analyzed and described.

  • 99% of the sequencing is completed by today.

  • Reference genome:

• Whole-genome shotgun approach: cloning and sequencing of DNA fragments from randomly cut DNA. Computers assemble very large number of overlapping short sequences into a single continuous sequence.

• Metagenomics: DNA from an entire community of a species is collected from an environment sample and sequenced.

Chapter 21.2: Bioinformatics used to analyze genomes and their functions.

• NCBI or National Center for Biotechnology Information has several resources, such as GenBank which houses sequence data for a wide variety of organisms.

• BLAST (Basic Local Alignment Search Tool): A widely used algorithm for comparing nucleotide or protein sequences to sequence databases in GenBank, helping to identify functional and evolutionary relationships.

• Gene Annotation → Consists of three evidences to identify a gene.

  • First, software is used to search for patterns which indicate genes, mRNA and sequences.

  • Second, clues are attempted to be obtained and function/purpose of potential gene. Software is also used to compare the code with already known DNA sequences.

  • Third, identities of genes using RNA-seq are confirmed.

• Proteomics: This field focuses on the large-scale study of proteins, particularly their functions and structures.

  • Proteome: entire set of proteins expressed by a cell or group of cells.

• Systems biology: Aims to model the dynamic behavior of whole biological systems.