The document outlines readings for different lectures in a biology course. Lecture 1 covers what will be taught including cellular and genomic diversity, model organisms, DNA, RNA, proteins, and more. Lecture 2 introduces cells and diversity, the tree of life, prokaryotic and eukaryotic cells, and model systems. Lecture 3 discusses molecular interactions, nucleic acids, and protein structure.
The document outlines readings for different lectures in a biology course. Lecture 1 covers what will be taught including cellular and genomic diversity, model organisms, DNA, RNA, proteins, and more. Lecture 2 introduces cells and diversity, the tree of life, prokaryotic and eukaryotic cells, and model systems. Lecture 3 discusses molecular interactions, nucleic acids, and protein structure.
The document outlines readings for different lectures in a biology course. Lecture 1 covers what will be taught including cellular and genomic diversity, model organisms, DNA, RNA, proteins, and more. Lecture 2 introduces cells and diversity, the tree of life, prokaryotic and eukaryotic cells, and model systems. Lecture 3 discusses molecular interactions, nucleic acids, and protein structure.
The document outlines readings for different lectures in a biology course. Lecture 1 covers what will be taught including cellular and genomic diversity, model organisms, DNA, RNA, proteins, and more. Lecture 2 introduces cells and diversity, the tree of life, prokaryotic and eukaryotic cells, and model systems. Lecture 3 discusses molecular interactions, nucleic acids, and protein structure.
Cellular and genomic diversity o Prokaryotes vs. Eukaryotes Diffs and sims General organisation Origins Model organisms: such as E. coli, C. elegans, M. musculus, Drosophila, Arabidopsis
The Molecules of Life:
General concepts about o DNA, RNA, proteins; their nomenclature, important structural components, bonds that hold them together
DNA - nuclear and organellar genomes (mitochondrial and chloroplast DNA vs. nuclear) - they perform different tasks and code for different proteins o mito and chloro have their own DNA because they were originally two organisms, one engulfed the other, and now they are endosymbiotic; one organism living inside another - chromosomes and chromatin o chromatin contains genes that modify gene expression in cell o how DNA is packaged and then unwrapped to be available for expression - DNA replication and repair - Genes and genomics
RNA - general characterisation of transcription o exons, introns, synthesising different kinds - RNA processing - Transcriptomics – all transcripts and proteins being expressed at one type (everything being transcribed – not [introns]****) Proteins - translation - post-translation processing - proteomics
LECTURE 2: Introduction to Cells and Diversity
Covered: Prokaryotic vs. Eukaryotic cells, Diversity and Model Systems, Origins, Introduction to Nucleic Acids
Tree of Life: shows how three domains of life (bacteria [eubacteria], archaea [archaebacteria], and eukaryotes) Some examples: - methanothermobacteria – anaerobic, low pH (acidic) conditions (cow stomach) - cyanobacteria – capable of photosynthesis, found in soil - methanococcus – found in hydrothermal vents at bottom of the ocean Chart has a scale showing 1 change/10 nucleotides (n) showing HOW different they are
Two Main Types of Cells:
Prokaryotes: no nuclei, single-celled organisms (e.g. eubacteria and archaea)
- has selectively permeable cell wall and plasma membrane - flagellum for motion (not all have this) - nucleoid: an irregularly shaped region in the cell w/ unprotected genetic material - ribosomes for protein synthesis in cytosol Eukaryotes: nuclei, single or multicellular, a lot larger w/ cytoskeletons for structural integrity (plants, fungi, animals, humans) - contains endomembranes, separating cell into functional compartments (organelles) - microtubules, centrosome w/ pair of centrioles - Nucleus w/ nuclear envelope containing DNA - Extracellular matrix and lysosome NOT found in plant cells - Mitochondria produce ATP - **Vacuoles do not exist in animal cells, are exclusive to plants
ENDOSYMBIOSIS: Origins of Mitochondria
1. Anaerobic ancestor engulfed an aerobic bacterium producing ATP from oxygen
2. Mitochondria escaped digestion, and was protected by the cell, providing energy, and containing its own DNA 3. Same story with chloroplasts and some algae Didinium: carnivorous, single-celled eukaryote that has cilia and shoots poison darts into its target and engulfs it Model Organisms: species that have been studied intensively for a long period of time and are models for deriving fundamental biological principles (e.g. drosophila) Attributes: - rapid development, short life cycles - small adult reproductive size (not an elephant) - readily available - tractability – ease of manipulation or modification - understandable genetics – simple organisms
A Model Prokaryote: E. coli – heterotrophic eubacterium in human gut
Synechocytis – free living phototrophic cyanobacterium
A Model Eukaryote: Yeast – minimal eukaryote model
Arabidopsis – a model flowering species Caenorhabditis elegans – nematode worm with 959 body cells Drosophila, mice, humans
Central Dogma: information flow in the cell
DNA ---------------- RNA -------------- Protein
Transcription Translation RNA synth protein synth
*Note DNA doesn’t actually turn into protein or RNA
Transcription Overview: DNA, RNA, and protein are all linear chains of information Information in nucleic acid sequence is translated into an amino acid sequence via a genetic code which is essentially universal (all cells have the same one) among all species. It is also degenerate and semi-conservative.
Genetic code: refers to the codons (a triplet of nucleotides) that correspond to a specific amino acid. It is degenerate because many different codons code for the same AA. CC“X” – Proline, UAA, UAG, UGA – stop codon, AUG – start codon
Consequences of degeneracy: 1. All 20 AAs are coded by the DNA 2. There are opportunities for incorporation of new codons or AAs 3. Base-pairing between codon and anti-codon structures occurs more easily 4. Random mutation resulting in AA changes are reduced, with less damage done
Nucleic Acids: the genetic material of a cell; an organism’s blueprints
Contains: - pentose sugar, a scaffold (railing) for bases - nitrogenous base, which varies (ACTG) - phosphate group, the backbone (1, 2 or 3 Ps) One monomer is all of these (a nucleotide) Purines: Adenine, Guanine Pyrimidines: Cytosine, Thymine, and Uracil
DNA: deoxyribose sugar used, ACTG bases (thymine has an extra methyl group) RNA: ribose sugar used, ACUG bases (ribose has an extra O)
Nucleic Acid Nomenclature
1. Nucleioside monophosphate (e.g. AMP – adenosine monophosphate) a. Sugar + base + 1P 2. Nucleioside diphosphate (e.g. ADP – adenosine diphosphate) a. Sugar + base + 2P 3. Nucleoside triphosphate (e.g. ATP – adenosine triphosphate) a. Sugar + base + 3P A nucleoside (no phosphates) is a base plus a sugar A nucleoTIDE is a base, sugar, and at least 1P e.g. a nucleotide is ADENINE, a nucleoside is ADENOSINE *others are: Guanidine, Cytodine, Thymidine, Uracine *nucleoside + sugar = -ina (cytodina)
DNA is a template for the three main types of RNA involved in transcription: messengerRNA (translation): protein transferRNA: transportation of amino acids for protein synthesis ribosomalRNA: part of the ribosome
Genome: all cells or organism’s genetic sequence
Transcriptome: all RNA species in cell Proteome: all protein in cell Interactome: all interactions in proteins or molecules Metabolome: all small molecule metabolites, or organic molecules (HORMONES) Phenome: complete set of phenotypes (all of the above)
LECTURE 3: Introduction to Nucleic Acids and Proteins
Molecular Interactions: interactions between individual molecules usually mediated by
noncovalent attractions. Phosphodiester bonds are those between phosphate and sugar in DNA. They are very stable and permanent Electrostatic attractions: ionic bonds within proteins due to high level of water in cell preventing charges from happening Van der Waals attractions: bringing together macromolecules Hydrophobic forces: hydrophobia causes molecules to fold into each other
Individually they are very weak, but can sum to generate strong binding between molecules
The amine end (N terminus) of a protein is like the 5’ end, and the carboxyl end is the 3’ (C-terminus)
Nucleic acid chains:
DNA is synthesised from dNTPs (deoxyribonucleoside triphosphates) RNA “ “ “ NTPs (ribonucleoside triphosphates)
Base Pairing: A-T make 2 H-bonds, C-G make 3 H-bonds
Structure: Major groove (larger segment) allows for access by proteins like modifying enzymes, with the bases. The strands are antiparallel, one is 5’-3’, the other is 3’-5’. At the 3’ end is a hydroxyl group (OH) off the sugar, and the 5’ has a phosphate (-PO4). High temperatures or pH will denature the DNA, and be unzipped. Bringing them back to normal conditions will cause them to reanneal and nucleotides bonds will reform due to complementary base pairing. This is a reversible process, the denaturation temperature is call Tm, and varies between species.
Polymerase Chain Reaction:
1. Heat to separate strands 2. Hybrization of primers pieces of DNA synthesised 15-20n complementary to gene wanted for study 3. Add DNA polymerase to add to existing fragments, making the sequence for gene of interest 4. DNA synthesised from primers
Introduction to Protein Structure:
Primary (sequence) Amino acid sequence Secondary (local folding) alpha helix, beta sheet Tertiary (long-range folding) 3-D structure Quaternary (multimeric organization) Multiple polypeptide chains Supramolecular (large scale assembly) Large scale protein assemblies ` Proteins are composed of amino acids The amino side-chains or “R” group is variable, and determines the type of AA 4 basic categories: basic (+ve), acidic (-ve), uncharged polar (hydrophilic), non-polar (hydrophobic) – these are consequences of the R groups
Cysteine can form Cys bridges across a chain of AAs
Mutational steps: # mutations between codons to turn them into a new AA # to get from proline to cysteine: 2, max 3
Groups with similar properties are clustered in the codon table, and have fewer mutational steps between them.
Order of AAs is important, i.e. Leu-Enkephalin, which modulates perception of pain –
causes sense of wellness when running for example. We always right N terminus to C terminus.
Alpha Helix: 3.6 AA per turn, girder like, steel frames, can create pores through membrane walls allowing certain molecules in based on the R groups Beta Sheet: runs antiparallel – H-bonds are formed between strands, like plywood, forming a wall
H-bonding in secondary structures
Which atoms are H-bonded? Carbonyl oxygen, amide hydrogen Alpha helices bond between 4 AAs apart within the same strand Beta sheets between AA in different strands
The alpha helices can have amphipathic qualities, wherein certain R groups will be on one side of the helix that are hydrophobic, and the other hydrophilic. Useful for when it crosses a pore leading into a cell - creates “supercoil”, or coiled coil, where polar goes with polar and v.v., coiled in again on its self
Quaternary structure: Haemoglobin formed from different subunits, 2 alphas and 2 betas, and each is a polypeptide
LECTURE 4: Genomes and Chromosomes
Neanderthal Genome Sequence:
- 65% known - DNA recovered from 38 000 year old bones in Croatia - Modern humans w/ non-African ancestral genomes share 1-4% DNA o Bone and skin are tougher (no clothes) o Ribs wider and smaller - Partial sequence analysis shows only 78 AA variants in protein coding genes that MIGHT impact function
Human Genome: 3 billion bps/genome, one maternal + one paternal = you
- ~25 000 genes spread across 23 chromosomes - Each group of 23 chromosomes from either parent is a genome .: Two genomes - Chromosome: an organised structure of DNA and protein - Genomes can be any size – mitochondria just 16.6 kb, chloroplast is 160 kb o So small because most coding is done by DNA, they no longer need genes to live solo, proteins made by DNA move to mitochondria - Prokaryote DNA is generally smaller, can sequence in hours - However, an amoeba has more kb than us – size doesn’t correlate to complexity!
1.5% of genome encodes protein – most is non-coding or redundant, 50% is repetitive
Transposons: - LINE: long interspersed nuclear elements - SINE: short “ “ “ - Retroviruses: stick their genetic material into your genome - Transposon fossils Repeated sequences: all of above plus simple sequence repeats, segmental duplications Unique sequences including: non-repetitive DNA neither in introns or codons, nothing to do with making RNA or proteins Genes: introns and protein coding regions
Packaging of DNA in Prokaryotic Cells:
DNA is condensed 1 000 fold, DNA + protein form the nucleoid Components that help packaging include: - +vely charged polyamines (proteins), react to the –ve DNA - NAP (nucleoid-associated proteins) such as H-NS, histone-like proteins HU, IHF, FIS - Supercoiling of DNA by topoisomerase, wind and unwinding DNA
Eukaryotic Cells: 6 billion bp/cell, around 2 metres, nucleus is 6 micrometres
Solution: packaged as chromosomes – karyotype: artificial array of paired chromosomes in numerical order, a diagnostic aid Chromatin: single, long, linear DNA and associated proteins - is packaged tightly, but DNA must be accessible for transcription, replication, repair - Dynamic Nucleosome (eukaryotic!!): basic structural unit of chromatin – think beads (proteins) on a string (DNA), linker + wrapper + core protein (histone), histone + wrap = nucleosome core, the wrapping requires ATP Components: - linker DNA 80 bps long - wrapper DNA around histones ~147 bps long - histones: small proteins +vely charged; stick well to DNA o four core histones: H2A, H2B, H3, H4, with a pair of each in octamer core o one linker histone in on linking part (H1), clips DNA in place on bead Heterochromatin: during M phase of division, includes mitotic and meiotic chromosomes, centromeres (centre of chromo.) and telomeric regions (end), one of Xs in females. In these locations gene expression is suppressed – no transcription! Euchromatin: (looser, more access) where genes tend to be expressed, covalent modification of histones, RNA polymerase and other allow for the switch between these two forms.
LECTURE 5: Introduction to DNA Replication
Replication is semiconservative – each daughter cell has a template strand and novel strand Always goes 5’ to 3’ ends, with three models: - Unidirectional growth from two starting points, e.g. linear viruses - Unidirectional growth of two strands from one starting point (some plasmids) - Bidirectional growth from one starting point, e.g. eukaryotes & bacteria Always starts at easy to open A-T rich segments (only 2 H-bonds) which are recognised by initiator proteins – TFII and the TATA box for example - single origin point: bacteria - multiple: eukaryotes with many origins of replication
Ex. Budding yeast with and without Autonomously replicating sequences (ARS) A plasmid vector is inserting with histidine producing gene, w/ and w/out ARS. Ones w/ ARS survive, ones w/out die because His gene is not replicated into future generations.
In bacteria: circular genomes are pulled apart and nucleotides added 5’-3’, looks like an eye opening up – points where parental strands are being pulled apart called replication forks. Leading strand: 5’-3’ going towards direction of separation Lagging strand: filled in based off the leading strand with Okazaki fragments. Between fragments are “nicks” that need to be covalently bonded; replication is towards 5’.
Ingredients for synthesis: Origin, ATP, primers, DNAases, DNA polymerase, accessory proteins 1. Origin of replication 2. Binding of initiator proteins: bind to origin, help helicase bind to initiator proteins, requires ATP 3. Unwinding by helicase: Has 6 subunits, unwinds in 5’-3’ along lagging strand template, requires ATP 4. Binding of single-strand binding proteins: keeps DNA separated to prevent H- bonding by binding ssDNA, preventing hairpins and kinks. 5. RNA primers made by primase: to begin DNA polymerase requires bound primase to synthesise an RNA primer with 3’ OH end to start binding to incoming nucleotides; goes 5’-3’ 6. DNA polymerase: attaches incoming dNTPs (deoxyribonucleoside triphosphate); has polymerizing and editing sections. 7. Sliding clamp holds polymerase onto DNA 8. Nick sealing between Okazaki fragments by DNA ligase: DNA repair system removes RNA primer and replaces it with the correct sequence
LECTURE 6: DNA Replication II
1. Leading strand synthesised continuously from single RNA primers
2. Lagging strand discontinuously from multiple primers 3. Okazaki fragment = RNA primer + DNA 4. Goes 5’-3’, primosome = helicase + primase 5. Helicase is located on the lagging strand
At ends of chromosome, the 5’ end, information (telomeres) is lost due to removal of the primers without a way to add more nucleotides behind the last primer. This is a problem for the lagging end – telomeres exist here to fold back into the same strand and protect coding information. Telomerase: generates G-rich ends, adding nucleotides to the 3’ end of lagging template - cancer cells have high levels of telomerase
Supercoiled in the same direction as DNA = + supercoils, opposite = - supercoils
Replication uses + supercoils
Stress released by Topoisomerase type I: breaks single-stranded DNA, allowing it to
rotate around the sugar phosphate group of one strand Topoisomerase type II: untangle and separates double stranded DNA (i.e. two plasmids that are separated), breaks double-stranded to allow one double-stranded helix to pass through the other
DNA Proofreading:
RNA polymerases have 1 in 10^4 error rate, DNA polymerases 1 in 10^9
Two proofreading methods: 1. 3’ to 5’ exonuclease: chews back misincorporated nucleotides – a literal backspace button – DNA polymerase engages in editing in a different site than its does polymerization 5’-3’ direction allows for efficient error correction and chain growth 2. Strand-directed mismatch repair: post-polymerase repair process, initiated by detection of distortion in the geometry of the helix by mismatched base pairs
- DNA can be damaged after synthesis by oxidation, radiation, heat or chemicals
- defects in repair mechanisms can cause breast and skin cancers 3. Base-excision repair: targets individual incorrect nucleotides
LECTURE 7: Transcription I
LECTURE 8: Transcription II
Introns are transcribed but not translated, exons are transcribed and may or may not be translated
M, t, and rRNAs code for protein
Three types of eukaryotic RNA polymerases (RNAPs) required for transcription:
1. RNA polymerase I – 5.8S, 18S, 28S RNA genes (larger S = larger RNA) 2. “ “ II – all protein coding genes (mRNA) 3. “ “ III – tRNA genes
RNAPs are complex structures with many subunits, some are common to all three, some resemble those of bacterial RNAPs – eukaryotic requires more proteins called “general transcription factors” because they have to deal with unravelling and coiling
Eu vs. bacterial RNAPs:
Eu requires proteins to help position them at promoter called transcription factors, which fulfill similar role to sigma subunit of bac RNAPs. Eu RNAPs also deal with chromosomal structure, nucleosomes, etc. - No introns in bacterial gene structure - Eukaryotic mRNA has guanine cap and poly-A tail.
Promoters: +1 = transcription starting point, “Upstream” are –ve #s, not transcribed, “downstream” is where RNAP starts making mRNA, which ARE transcribed.
1. TATA box: is highly conserved, 25-36 bp upstream of start point, positions
RNAP II 2. TFII is the transcription factor for RNAP II, TBP is TATA binding protein
Initiation of transcription: RNAP II ACTIVATED THROUGH
PHOSPHORYLATION**** 1. TBP is a subunit of TFIID in highly transcribed genes, binds to TATA box, bending and distorting the DNA to shape it better for RNAP II 2. TFIIH: large, 9 subunits; ‘H’ is for helicase, opens up DNA strands and requires ATP to pry apart DNA strands at start point 3. TFIIB: binds to BRE element helps position RNAP II 4. CTD: carboxyterminal domain 5. All of above are “Transcription initiation complex” 6. Complex begins doing “abortive” (stop and start) transcription 7. TFIIH comes in, “kissing”, then makes it transcribe at high speeds; also phosphorylates the 5th Serine of CT tail, so that transcription can begin a. Histone modifying enzyme: covalently modifies the parts
RNA Processing: 1. Add 5’ pre-mRNA cap – removes 1 P, adds GMP (guanidine monophosphate), and adds methyl group to G base guanine becomes first ribose 2. Guanine cap is added to the 5’ end of transcript, making a 5’-5’ linkage (BACKWARDS), enzymes involved: phosphatase, guanyl transferase, and methyl transferase. 3. Prevents exonucleases from chewing the transcript
Pre-mRNA splicing reaction: (splicing proteins remove introns & some exons) 1. Adenosine attacks the 5’ splice site 2. 3’ of one exon reacts w/ 5’ of next exon to release introns – they merge 3. The introns forms a lasso shape around adenosine called LARIAT 4. Process requires the 2’ OH group, .: cannot happen in DNA (deoxyribose)
RNA and protein interactions for splicing: snRNA = small nuclear RNA - snRNPs = snRNAs + proteins - Pre-mRNA splicing is performed by the spliceosome, ATP-dependent, binds to many nucleotide sequences
LECTURE 9: Translation
- codons are read as triplets
- DNA encodes for all 20 AAs - There is redundancy – multiple codons for most AAs - Reading frames define the AA sequence o If you move even one nucleotide over, it changes the entire AA chain
tRNA: ~80 nucleotides, each triplet in the anticodon region is antiparallel and complements the codon of the mRNA - some tRNA can recognise more than one codon
Ensuring fidelity: the right tRNA has to have the right AA on it
- tRNA synthetase puts the right AA on the right tRNA o similar to DNA polymerase, uses a backspace method for chopping incorrect units by hydrolytic editing o identifies tRNA anticodon nucleotides, recognises nucleotide sequence of acceptor stem, reads nucleotide information at additional position on the tRNA - complementary base pairing
Ribosomes: Eukaryotic is larger than prokaryotic
- located on endoplasmic reticulum (eukaryotes only – pros don’t have ER) and cytosol
Since protein synthesis is slow, polysomes, or polyribosomes occur – many ribosomes
spaced every 80 nucleotides are reading the mRNA and making the requisite proteins
Quality Control: GTP w/ EF-Tu (elongation factors) I escort aminoacyl-tRNA to
ribosome and checks for correct AA and anticodon. Also holds tRNA away from the P- site to prevent premature peptide bond from forming. Later, EF-Gmoves the small ribosomal unit (bottom one) quicker so the codon nucleotides don’t lag behind.
Elongation factors: - speed up and make more efficient the process of protein synthesis - mediated by EF-Tu, and GTP hydrolysis - EF-Tu binds aminoacyl-tRNA - EF-G helps ratchet forward one codon
Prokaryote ribosome binding site are also called “Shine-Dalgarno sequences”, and have multiple initiation sites
Termination of translation: Human translation release factor
- molecular mimicry – looks like a tRNA, but is a protein
Proteins fold as they exit the exit of the ribosome, happens at the same time as translation When things go awry, protein chaperones (Hsp60, Hsp70) catalyse the proteins to make them fold properly – if that doesn’t work, a protease destroys the useless protein
H. Denis Burges, Keith A. Jones (Auth.), H. D. Burges (Eds.) - Formulation of Microbial Biopesticides - Beneficial Microorganisms, Nematodes and Seed Treatments-Springer Netherlands (1998)
H. Denis Burges, Keith A. Jones (Auth.), H. D. Burges (Eds.) - Formulation of Microbial Biopesticides - Beneficial Microorganisms, Nematodes and Seed Treatments-Springer Netherlands (1998)
