Typical Prokaryotic Cell - Escherichia coli Single Cell Organism – organism on the face of the earth that we know the most about; best studied and understood organism (model that allows us to understand the workings of a prokaryote) Binary Fission – division of the cell which occurs in a nutrient rich environment every 20 minutes; in the entire earth, there are 1030 of these organisms Organisms undergo self-replication – increases in size and divides, then the same process repeats In a day, ther

LECTURE 1 Explosive Bacterial Growth -video is a bacterial cell undergoing cell division -self-replication: organism was able to take nutrients from environment and divide – bacteria have qualities of life, interacts with environment Cell Division by Binary Fission -takes nutrient from environment -increases size -replicates DNA -lays down cell wall -from one cell to two Generation time = 20 minutes One day – 72 cell divisions Total number of cells; N = 272 = 4.7 x 1021 cells @ 865 fg / cells, 4

Mostly unicellular, micro-organisms Even multi-cellular organisms are generated from single cells Characteristics of life Growth Metabolism Movement Response to stimuli-communications (even cells communicate) Reproduction The Cell Smallest unit of life All living things are made up of cells New cells arise only from pre-existing cells Cell and molecular biology + principles of evolution universal features All cells store their hereditary information in the same linear chemical code (DNA) (A) B

Central Dogma of Molecular Biology - Flow of Information DNA is a molecule that contains information – contains discreet portion of information, not a big, long piece of information but they are segments They are transcribed – creating a copy of information that serves as a template for protein synthesis Information encoded in DNA is information to make a protein – specifically encoding the series of amino acids At the molecular level, these functions are complex and more complicated All organis

Lecture 2 What is a Cell? Not only is it the fundamental unit of life, but to define a cell in an empirical, biochemical definition: A lipid-membrane-bound unit filled with a concentrated and organized aqueous solution containing -proteins -nucleic acids -carbohydrates -lipids -variety of organic compounds -inorganic ions -organelles (not all cells) -water That collectively displaying the 5 characteristics of life Cells vary greatly in composition, size, and function *Central Dogma is the unifyi

Nucleotide Structure & DNA This nucleotide is involved in a number of processes – it delivers energy to drive biochemical reactions It is also involved in forming part of the structure of DNA – not quite ATP but it is deoxy-ATP that is incorporated into the DNA We can see that a simple structure can be put together in a spiral shape molecule with repetitive structure to it Polymerization: 3’ to 5’ Phosphodiester bonds DNA/RNA: Linear polymer of nucleotides The substrate for the reaction is a nuc

Chemical Systems Living Creatures Components, environments, structure function 70% water in a cell, , polar molecule = 96.5% of organism's weight How do these interact together? Covalent bonds - stronger because of the way they share their electrons Non-covalent bonds - weaker bond interactions between molecules TYPES of non-covalent bonds: Electrostatic ionic bonds between components with charges (partial positive/negative) enhances interactions between components Hydrogen bonds enha

LECTURE 3 Central Dogma of Molecular Biology – Flow of Information -important because present in all living cells -common feature that distinguishes every living organism Genetic Info is Stored in DNA -Streptococcus pneumoniae is a bacterial pathogen that creates bacterial pneumonia in mammals, has coating around outer surface – sheath – gives cells smooth texture -rough appearance lacks surrounding sheath -Smooth (S) strain causes pneumonia -Rough (R) strain does not cause pneumonia First Exper

Levels of Protein Structure Primary (1) Structure The amino acid sequence of polypeptide chain The linear sequence of protein sequence – N-terminal to C-terminal Typically finding proteins ranging in size from 50 amino acids to over 2000 amino acids Single letter abbreviations for the amino acids as well – for instance, cysteine is C or cys (three letter abbreviation) Chemical properties of amino acids that give the biological and chemical properties of proteins The primary sequence of amino aci

Initiation The origin of replication (Ori C)' is N245 base pairs long. DNA-A binding region and A-T rich region. For replication to begin, DNA needs to separate at the A-T rich region. This happens easilty because A-T are double bond (H-bond) DNA-A is a homotetramer - 4 identical monomers/subunits DNA-A proteins bind to the DNA-A binding site strand separation SSB- Single stranded binding proteins, bind to ssDNA to prevent the separated strands from re-annealing (Homotetramer). DNA-B: Helicase

Nucleic Acids - DNA and RNA (also energy molecules...) Nucleotides structure Sugar -- glycosydic bonds-- Nitrogenous base -- Phospho and hydro bonds (good source of energy) -- # of Phosphates Nucleoside Phosphate - # Phosphates Name ( 3 phosphates = triphosphate) Purine - Nitrogen on 1, 3, 7, 9 (2 Rings) Pyrimidine - - Nitrogen on 1, 3 (1 Ring) Sugar - Hydroxylic group - Present - Rybonucleotide on - Absent = Deoxyribose When both hydroxyls are missing ( C-2', 3') = Dideoxyribose Pho

LECTURE 4 DNA/RNA – Linear Polymer of Nucleotides -deciphering DNA structure Rosalyn Franklin’s X-Ray Diffraction Image of DNA -Franklin and Wilkins analyzed DNA by bombarding DNA crystals with x-rays. Their helical image analysis yielded two numbers that sparked interest – 3.4 nm and 0.34 nm, which we now know that these numbers represent the distance between nucleotide base pairs and spatial distance between one complete helical turn and another in the molecule DNA/RNA – Linear Polymer of Nucl

RNA Polymerase has 5 subunits - Alpha (x2) Beta Beta' Omega For RNA Pol to bind to DNA, it must be in complex with the sigma factor. Different sigma factors are used for transcription of different genes. Holo-enzymes - All RNA Pol subunits together + sigma factor Promoter region has 3 binding sites. 10 - Here sigma factor binds. 35 - Here also sigma factor binds. Up-element - 10, 35, and 50 base pairs upstream of transcription start site. 2 Alpha subunits bind here. Once sigma factor is bound,

Composition of Cells Hydrogen Carbon Common to all biological molecules Nitrogen Oxygen Phosphorus Sulfur Carbon and sulphur have 4 valence electrons, but carbon can form 4 stronger and stable bonds than sulfur. This is because the 4 valence electrons carbon have are closer to the nucleus.When carbon combines with other carbons, it forms strong skeleton structures composed of linear, branched, or ringed groups of carbons. Side groups of biological molecules are central to the function of a molec

DNA/Protein Interactions EcoR1 – a protein that is capable of binding DNA, not randomly but at a specific nucleotide sequence and it is a protein catalyst – reaction that the protein catalyzes is phosphodiester bonds It is a homodimer – made up of two identical subunits; catalytic activity requires cofactor which is magnesium Presence of magnesium will cleave the phosphodiester bond in two places, breaking DNA apart Nucleases - (Proteins) EcoR1 is a part of this group Enzymes that cleave phospho

LECTURE 5 Proteins and their functions Proteins participate in a diverse array of cellular functions -enzymes -structural components -signaling -receptors -motor proteins -transport -gene regulation -immune system antibodies -storage Functions are related to their unique structure and composition -3D structure and chemical reactivity contributes to defining protein function -proteins are the most structurally complex molecules Proteins: linear, unbranched polymer of amino acids linked by peptide

6.1.1 - 6.1.3: Introduction to Protein Trafficking The Protein Trafficking Machinery Proteins are machines that carry out virtually every function in the cell and are therefore required at every cellular location. The route from initial translation to final destination is highly regulated. Protein production, sorting, trafficking, and targeting are so fundamental to cellular function that the cellular machinery that performs these functions are usually the largest and most obvious components of

UTR’s flank the coding sequences and have specific functions Two terminators in prokaryotic genes – one that terminates translation and one that terminates transcription Prokaryotic genes lack introns Transcription with the end product of an RNA molecule (single stranded polynucleotides made up on nucleotides C, A, U and G) Transcription involves a complicated enzyme known as RNA polymerase which is an enzyme catalyst that we can describe the biochemical reaction that it participates in One of t

Replication Transcription Translation Central Dogma - DNA DNA RNA Protein RNA Synthesis Protein Synthesis Gene Sequences have many functions, the definition is less accurate. A segment of DNA that encodes information for a protein or functional RNA as a linear sequence of nucleotides. Each gene has a different product (a double strand has different parts - genes within it.) Different genes are transcribed/translated in different w

Lecture 6 Amino Acid Sequence Influences How A Protein Folds -folding of proteins take on physical and chemical interactions between backbone and side chains -2 cysteines can form covalent, disulfide bonds becoming cystine (by oxidation) and holds polypeptides together – a molecular staple – in close proximity, important for 3D structure of protein’s shape, high degree of stability Non-covalent interactions affect protein folding -electrical charge, weaker electrostatic attraction and hydrophob

Nucleic Acid Polymers and Transcription The only strand RNA that can continue to interact with is the 3' 5' strand, this is the one being read in RNA synthesis: 5' 3' is the directionality of the RNA strand. DNA - Coding strand 5' 3', because it is almost identical to the synthesized RNA strand. Non-coding strand - 3' 5' dRNA transcription - 5' 3' synthesized direction template strand Coding - Non-template - (+) Strand Non - Coding - Template - (-) Strand mRNA transcript - Sense Upstream

Sigma factor allows them to identify where the protein is located on the DNA Core enzyme contains catalytic activity of the enzyme – beta subunit catalyzes It is the sigma factor that is the regulatory component and recognizes the promoter General functions include Alpha recognizes the upstream element in DNA Beta has the polymerase activity which includes chain initiation and elongation Beta prime is involved in binding DNA and stabilizes the structure on the template of DNA Omega is a molecula

LECTURE 7 Protein Structure - 3° Secondary Two views of the 3D arrangement of rattus norvegicus carbonic anhydrase 2 that catalyzes -enzyme is crucial for respiratory gas exchange, helps mobilize CO2 -active site is where reaction happens The active site Zn coordinated by 3 HIS residues rattus norvegicus carbonic anhydrase 2 -brings histamine residues together in close proximity to coordinate zinc atom -absence of zinc, enzyme is non-functional -importance of protein folding: allows for formati

Transcription of a Eukaryotic Gene II Transcription Initiation Complex Eubacteria - core RNA polymerase (5 subunits) requires sigma factor to recognize -10 & -35 regions of the promoter Eukaryotes - ‘NA polymerase II (5+7 subunits) also requires ‘help’ to recognize the promoter: requires general transcription factors Not as straightforward as eubacteria, there are 7 additional subunits which they add extra complexity RNA II polymerase cannot recognize the promoter and requires HELPERS – call

Transcription Process Divided into 3 main stages - Initiation, Elongation, Termination RNA polymerase is needed to recognize the promoter region - Which involves amino acids (in RNA Pol) recognizing nucleotides in the DNA. Recognition process is essential by which the amino acid sequence and nucleotides have been conserved for evolutionary times. They have similar sequences in different species. With the help of factor, helps (RNA Pol) to bind and align To transcribe in the correct place To tra

Lecture 8 Genome: cells entire library of genetic information (DNA) Eukaryotes DNA in nucleus, mitochondria and chloroplasts – segregated Prokaryotes Nucleoid – integrated + plasmids (extrachromosomal DNA: self-replicate independently from the chromosomal DNA and carry genetic info which is important for survival of cell under specific conditions, most important ones are antibiotic resistant genes) Gene: segment of DNA which controls discrete hereditary trait, characteristic or phenotype -observ

Process that involves the synthesis of a linear sequence of amino acids – proteins themselves are made up of a limited number of 20 L-amino acids Protein synthesis process has a directionality to it –very discreet, one step at a time process, one peptide at a time is formed Protein begins synthesis at N-terminus, guided by the mRNA which is translated from the 5’ end to the 3’ end of RNA Leads synthesis of protein from N-terminus to C-terminus Once the process is finished, there is post-translat

Regulation is in groups in the same pathway. Eukaryotes - Transcription 1) Only Monocistronic - The way messages come in. 2) 3 types of RNA Polymerase to produce different types of RNA. 3) More complicated than prokaryotes. Compartmentilazation Exons / Introns (Why Introns in the first place?) DNA organization: tightly condensed chromatin with many proteins. Bacterial DNA has far fewer proteins. More players - different molecules and subunits The more complex - the more need of regulation. Havin

Lecture 9 Genes Are Not Easy to Find in The Genome They have a… “start” (promotor) “stop” (terminator) region of DNA between “start” and “stop” part of gene is transcribed to RNA contain “open reading frame”, an ORF – frame that codes for protein itself Prokaryotic Gene -usually lacks introns -gene: all nucleotide sequences required for both transcription, translation, and expression of that gene, not only region transcribed but also other regulatory or control factors Includes: Promoter: region

Upstream regulatory sequences that affect the level of transcription. They are very far from initiation site, but they are recognized by certain proteins (enhancer and activator proteins). Once the activator protein recognizes the upstream area, the DNA loops around and it is able to interact with the complex (not directly, but through the mediator). Mediator connects between enhancers and transcription initiation site. This extra protein is an extra regulatory effect by enhancing/depressing th

Detailed tRNA Secondary Structure Variable loop – variety of nucleotides (3-21 bases) T stem and acceptor step – acceptor stem has a conserved nucleotide sequence (5’ CCA) – 100% conserved for every tRNA Every amino acid is added to the tRNA molecule through the acceptor stem One interface where it interacts with RNA (anticodon loop) The Wobble Hypothesis Anticodon-codon process does not necessarily involve obedience to the Watson-Crick pairing Pairing 3 is the wobble position – non-Watson and

Lecture 10 Transcription – Prokaryotes Monocistronic vs Polycistronic Message Monocistronic: nucleotide sequence codes for single polypeptide Polycistronic: arises when complex has single promoter region that contains two or more genes -way in which bacterial cells can ensure that genes for related function and transcribed and translated at same time RNA Polymerase -catalyzes synthesis of RNA -composed of 5 subunits -two identical regions – alpha 1 and 2 -beta region, beta’, and omega -sigma fac

Synthesis and subunit assembly in nucleus Massive amount of machinery that appears to be a small job – the machinery is needed to read the mRNA and incorporate the correct amino acid according to code on mRNA – complex! Typical cell contains millions of ribosomes – free in cytoplasm or attached to ER Ribosome Location Ribosomes inhabit a pool in the cytoplasm – every large subunit is NOT always associated with the small subunit – they associate and dissociate A common pool for cytosol and one f

TFIIH Phosphorilates with its kinase activity (before it used its helicase activity). On the correct phosporelation RNA Pol is going to escape the full PIC, and dissociates from most pre-initiation factors because it is correctly and tightly bounded to DNA. TFIIH stays. CTD - Convenient for it to extend outward, hard to bring more compound into the complex. It has much variability and variety of consequences, and many different proteins can bind there. Acts like a loading dock for proteins. In P

Lecture 11 Transcription -prokaryotes-directly make mRNA molecule and protein simultaneously, transcription and translation occur same place same time -eukaryotes-more complex, compartmentalized -introns and exons -primary transcript is hnRNA (heterogenous nuclear RNA), not the final transcript because needs to undergo biochemical processing reactions to become mature mRNA -modify 5’ end, modify 3’ end, excise introns, ligate exons into single mRNA -mRNA exported into cytoplasm and translation o

Function of rRNAs? 16S rRNA 3’ end – translation initiation + S1 & S2 proteins Stabilizing correct codon-anticodon pairing – participates in the recognition and stabilizing components Structural role – scaffold for protein binding 23S rRNA Catalytic subunit as well; involved in proton transfer during the peptide bond formation Exit tunnel from interior core to the external ribosomal environment that allows growing polypeptide to exit from the ribosome May contain up to millions of ribosomes in e

Polysystronic - One transcript that multiple proteins are transcribed. Splicing - Introns are removed and exons join and the result is 1 protein transcribed. What is the sequence in coding? 5' 3' on the coding strand RNA sequence 5" ACUUAC 3' DNA sequence 5' ---A-C-T-T-A-C--- 3' Nontemplate, Coding 3' ------------------ 5' Template, Non-coding

Lecture 12 Transcription Factor SP1 – An Example -SP1 is a DNA binding protein that interacts at the GC Box (proximal promoter) -member of a large protein family called Sp / KFL -composed of 785 amino acid with molecular weight of 81 kDa -contains 3 zinc finger DNA binding motifs -involved in cell growth, immune response, chromatin re-modelling -25 other known protein / protein interactors -SP1 – GC box interaction -SP1 binding affinity modified by phosphorylation, acetylation -SP1 – GC box – am

Translation - RNA Nucleic acid Protein Amino Acid Involves over 100 macromolecules mRNA - Messenger RNA: codes for proteins tRNA - Transfer RNA; Adapter between mRNA and aa during protein synthesis. rRNA - Ribosomal RNA: forms core of ribosome's structure + catalayzes protein synthesis. Y Bases 20 amino acids (A = met): 1 nucleotide (singlet) 4 amino acids (AU = met): 2 nucleotides (doublet) 42 = 16 amino acids (AUG = met): 3 nucleotides (triplet) 43 = 64 amino acids Smallest combina

Cells have a remarkable ability to copy molecules of DNA with specific sequences Cells have used DNA replication to ensure that copies of DNA are passed from one generation to another Only recently have chemists and biologists have been able to mimic what a cell actually does and to generate large amounts of DNA Kary Mullis developed PCR in 1983 and was awarded the 1993 Nobel prize in chemistry – Dancing Naked in a Mind Field was his book; he was an eccentric character! Polymerase Chain Reaction

Lecture 13 Transcription – Eukaryotes – Elongation RNA polymerase II escapes in elongation phase RNA Processing -sequential addition of ribonucleotides to the growing RNA molecule (nascent RNA molecule – within transcription complex) and formed/synthesized in the moment -is located near site where nascent RNA emerges -release of RNA polymerase II from PIC -has binding sites for several enzymes -involved in all primary (hnRNA) transcript processing -5' capping, splicing, and 3' poly A tail -acti

Eukaryotic Initiation All protein synthesis need to start by establishing an initiation complex – this complex in our eukaryotic system involves eukaryotic initiation factors and these initiation factors are differentiated by the prokaryotic counterparts with the little e (eIF-1A, eIF-3) 43S initiation complex in eukaryotes eIF1 and eIF3 that help to pry apart the large subunit from the small subunit and establish this component where the charged tRNA can establish a connection It is not well de

Lecture 14 Elongation – Excision and Splicing -conserved nucleotide – branch point – the intron branch point is always an “A” (adenosine) Mechanism for intron removal -phosphodiester bond broken between exon 1 and intron -intron rolls back onto itself to form covalent bond at branch point -next intron – exon junction is broken -two exons are joined together + product of lariat shaped RNA molecule Excision and Splicing – Target and mechanism at exon / intron junctions -intron branch point is alw

Wobble base tRNA base pairs with the mRNA sequence (numbered before) Allowed to bind to different nucleotides, because the base pairing is not perfect and this occurs on the 3rd position of the codon and the 1st position of the anti-codon. This is why the bass are numbered (5' - 3') Why it is not true that 61 codons require 61 amino acids? There are more than one codon that can correspond with one a.a. (Degeneracy) - More than one tRNA coding for the same a.a. and wobble base mechanism. Aminoac

50s Initiation Complex In prokaryotes, the shine delgarno sequence is before the start codon. Eukaryotic tRNA also has a methionine (Met) elF2, GTP... but the CAP and the poly-A tail play a role of recognition of the mRNA as a mature RNA. IFYE and IFYG can recognize and mediate interaction of cap and tail at the same time Looping mechanism-efficient Initiator tRNA moves along RNA searching for the first AUG assisted by Koza K consensus sequence

Asymmetric – particular piece of DNA will be synthesized in both directions Semi-discontinuous – leading strand is synthesized continuously while lagging strand is synthesized discontinuously, resulting in semi-discontinuity Primer that eventually gets put down which is RNA utilizing DNA as a template Should a wrong nucleotide be incorporated, there’s a mechanism that will remove and replace it DNA polymerase is self-correcting DNA polymerases can remove the wrong nucleotide and the correct one

Gametes are considered haploid, one set of genomic content Cell division - The genomic material should be duplicated - Daughter strands DNA Replication Semi conservative - Starting from a DNA Duplex Polymerization - 5' 3' adding to the 3' Origins of Replication (ORI) In a chromosome, we have multiple origins of replication. One replication fork on each side of the replication bubble. Bi-directional movement in the bubble. Semi discontinuous Continuous - Leading strand Discontinuous - Laggin

Direct source of energy for polymerization would be missing if DNA strands were synthesized in 3’ to 5’ direction – important part of DNA complex – improves the replication Errors occur at 109 base pairs The replication bubble is being stabilized by SSBs constantly – single stranded DNA strand exposed will be in complex with single stranded binding proteins Other components that are a part of this are clamp loader with TauC linker region – this is a complicated protein complex that allows for lo