Molecules and Cells notes

Intro to cells

Cells can be solitary or part of a community Individual cells of multicellular organisms are specialized and have intricate functions and forms

and communications Cells are broken down into prokaryotes and eukaryotes Prokaryotes

o Can be many shapes Spherical Rod Corkscrew/spiral

o Reproduces quickly (about 20 min)o Most diverse cells

Explore many habitats that eukaryotes cannot tolerateo Broken down into bacteria and archaea

The archaea are the prokaryotic cells that live in extreme places (extremophiles) Eukaryotic cells

o Larger defined membrane bound organelles with different functionso Cytoskeleton that directs movement

Model organismso Used by researchers because a part of them is similar to us

Molecules

Covalent bonds=sharing of electronso Nonpolar covalent-equalo Polar covalent-unequal and partial charges

Noncovalent bondso Ionic o No sharing

Life itself is carbon basedo Carbon forms 4 covalent bonds to 4 different atoms

Hydrogen bonds are vital to lifeo Found in watero Found in DNAo Stabilize the shape of lysozymes

Hydrophilic interactionso Molecules that tend to interact with water via hydrogen bondo Tend to be charged or polar

Hydrophobic interactions

o Uncharged nonpolar molecules that do not interact with water Van der waal interactions

o Weaker than H-bondso The dipole interactions are very weak and transiento Distance dependant

pH=-log[H+]o concentration of hydronium and hydroxide ionso more hydronium more acidico more hydroxide more basico 0-7 acidico 7-14 basico pH of 7.4 is blood…slightly basic

buffers can act as acids or baseso amino acids

4 elements make up 99% of humanso H, C, N, O

7 make up .9%o Na, Mg, K, Ca, P, S, Cl

4 major families of organic compundso Sugarso Fatty acidso Amino acidso Nucleotides

Proteins are the most abundant and versatile of the macromolecules Macromolecules are constructed by polymerization via condensation reactions where a

molecule of water is given off Break downadd water

Amino Acids

Amino groupo Acts as a base

Carboxylic acid groupo Acid

Alpha hydrogen off the main carbon Side chain (R group)

o The only thing that differs in each of the 20 AA It exists in an L and a D form (enantiomers)

o Our bodies only use the L form Three groups

o Polar

o Nonpolaro Electrically charged (ionic)

Functionso Protectiono Transporto Receptorso Contractileo And many more

R groups differ in shape, size, and reactivity with water PKA

o It is the pH where it is half protonated and half deprotonatedo Only the R group matters (amino and carboxy groups always dissociate)

Proteins themselves are made up of clusters of amino acids Connected by a peptide bond

o Rigid and not very flexible at the bond point Dehydration synthesis Hydrolysis (break down) Repeat of NCC

o The beginning amino and the end carboxy are the only charged part of the backbone N-teminus and C-terminus 5 levels of protein structure

o Primary Amino acid sequence

o Secondary Folding alpha helix and beta pleated sheet Stabilized by hydrogen bonds

o Tertiary Folding as the unit All the secondary structures Stabilized by:

Disulfide bonds H-bonds Covalent bonds Van der waals

All these bonds are between side chainso Quaternary

Protein has multiple tertiary structureso Domain

Domains are protein substructures that can fold intependently into compact, stable structures

Different domains have different functions

Part of a larger proteins Specific function Function can operate independently of the rest of the protein

Protein foldingo Protein folding is often spontaneous

Doesn’t need any helpo Some proteins need molecular chaperones which give them an environment to foldo Denature

Proteins unfold due usually to heat or pH changes Disulfide bonds are hardest to break Misfolded proteins cause human diseases

Prions β-mercaptoethanol breaks disulfide bonds SDS (sodium dodecyl sulfate) totally denatures a protein

Lipid, Membranes, first cells

Plasma membrane is a lipid bilaryero Separates the cell from the outside environmento Chemical reactions are more efficiento Selective barriero Localized specific functions

Two types of lipidso Fatty acids

Hydrocarbon tailo Isoprene

Lipids have a major hydrocarbon component and are mostly nonpolar and hydrophobic Steroids

o Ring structure Phospholipids

o Choline, Phosphate, glycerol, 2 fatty acid tails Ester linkage between the glycerol and the fatty acid

o Amphipathatic Polar and nonpolar component

o Hydrophilic heado Hydrophobic tails

Kinks in the fatty acid chains caused by cis double bondso Creates more space, allows the membrane to be more fluido Found in cold environments

Shorter tails also increase mobilityo Less forces

Temperature increases, fluidity increases

o Vice versa Very hot conditions? Not many kinks and the chains are longer

o Vice versa for cold Long and saturated=less permeable Short and unsaturated=more permeable Amphipathatic molecules form micelles or bilayers depending on the shape

o Cone shapemicelleso Cylinderbilaryer

Permeabilityo Small nonpolar molecules get in easy (hydrophobic)o Small uncharged polar molecules get ino Large uncharged polar don’t get ino Any ions cant get in (charged)

Eukaryotic cells add cholesterol to change permeabilityo Disrupts van der waal forceso Reduces membrane fluidity

Fluid mosaic modelo Proteins span across the membrane

Predictions of the fluid mosaic modelo Membranes are made up of 2 layers with hydrophobic chains on the interior and polar

head groups exposed to the exterioro Mobility of lipids is allowed only lateral and rotational and never a flip flopo Bilayers are asymmetric in nature

Differ in lipid concentrationo Membrane proteins and either integral or peripheral

RBC it is no cytoplasm Can see the bilayer asymmetry through an antibody reaction with both sides Phospholipase C

o Degrades the phospholipid membrane Use a detergent to gain access to the inside Can see the mobility of the phospholipids

o Fusion experimento Tagging with florescence

Raise the tempmore mobility Types of membrane proteins

o Transmembrane and membrane associated Transmembrane –all across Membrane associated-only one side Lipid linked-inside or outside, linked to phospholipids Protein attached-one transmembrane protein and another attached protein

If there are 18-21 nonpolar proteins in a row, there is most likely a transmembrane domain

o Usually an alpha helix Most proteins have a minimum of 3 domains Passive vs active transport

o Passive is diffusing without energy down a concentration gradiento Active requires an input of energy and against a concentration gradient

Linear graph=passive transport Passive across membranes

o Ions (need a channel)o Aquaporins (allow water)o Carrier proteins

Ion channelso Highly specialized for only allowing a certain ion ino Concentrationo Or chargeo Gated or passive channels

Saturation rate with transporto Eventually reach a maximum and it levels offo Can only diffuse so fast

Inside the Cell

Prokaryoteso Not too many organelles (no membranous ones)o Ribosomeso Circular DNA

In a nucleoid regiono Cytoskeleton

Plays a role in cell division, shape determination, and polarity determinationo Cell wall

Made of peptidoglycan Gram positive

o Thick peptidoglycan wall Gram negative

o 2 membranes Peptidoglycan and lipopolysaccharide and protein

membrane Releases endotoxins Harder to defend

o Plasmids Able to be shared with other prokaryotes Circular

Eukaryotic cell wallso Fungi, algae, plantso Stiff cell wall for cellular supporto Semi permeable

Permits small molecules and small proteins (30-60 kda)o Plants and algae have a cell wall made of celluloseo Fungi=chitin cell wall

Eukaryotic cells have membrane bound organelleso Nucleuso ERo Golgio Peroxisomes

Secretory cellso Secrete a product (can be good or bad)o Make things

Highly specializedo Produces material and secrete into a duct or the blood

Exocrine gland=duct Endocrine gland = blood Secretion is regulated Products are released from secretory granules

Nucleuso Surrounded by a double membrane nuclear envelopeo Nucleolus=site of RNA/ ribosome assembleo Fibrous proteins form a lattice

Nuclear lamina Structure and shape of the nucleus

o Function Information, storage, and processing Contains the cells chromosomes (code) Ribosomal RNA synthesis

o Nuclear membrane is an extension of the rough ERo Things can get through a nuclear pore complex

Regulates proteins going in and out Selective based on size

o Nucleus has a distinct set of proteins that work with ito Each protein has a targeting sequence within the primary sequence

Molecular “zip code”o Nuclear localization signal (NLS)

17 AA signal that tells a protein it belongs in the nucleus Rough ER

o Network of membrane bound tubes and sacs studded with ribosomeso Interior is the lumeno Rough ER continuous with nuclear envelopeo Hugs nucleus tightlyo Function

Ribosomes in rough ER synthesizes proteins Proteins are folded and processed and assembled in the lumen Proteins are glycosylated in the ER lumen Begin protein modification

Majority occurs in the golgi Smooth ER

o Rough ER without ribosomeso Enzymes within smooth ER produces fatty acids and phospholipids or breaks down

poisonous lipidso Calcium ion reservoir

Golgio Golgi apparatus is formed by a series of flat membranous sacs called cisternaeo Function

Golgi processes, sorts, and ships proteins synthesized in the rough ER Also site of lipid synthesis Membranous vesicles carry products to destination (either inside the cell or out) Carried in vesicles to the golgi too

o 3 faces Cis golgi is closest to the nucleus Medial golgi Trans golgi-closest to the PM

o The proteins can have destinations Resident in the cis golgi Continue to go on to inside or outside the cell

o Proteins in the lumen are dumped outside the cello Membrane sequence can allow a protein to be transmembraneo Destinations?

Lysosomes Plasma membrane Secretory vesicles

o Add oligosaccharides to proteins that are secreted out of the cell Mitochondria

o 2 membranes Outer Inner-cristae

Folded

o Has its own DNA and ribosomes Supports the belief that they used to be cells

o Function ATP production

Peroxisomeso Globular organelles bound by a single membraneo Function

Center of oxidation reactions (contains them) Catabolize long chain fatty acids, branched fatty acids, polyamines, and creates

plasmolagenso Specialized peroxisomes in plants called glyoxysomes

Packed with enzymes that oxidize fats to be used in energy storage Lysosomes

o Membrane bound structures containing approximately 40 different digestive enzymeso Found in animal cellso Function

Digestion Waste processing 40 different enzymes break down

Nucleic acids Carbs Lipids Proteins

o Working pH is 5.0 Has a pump to maintain a low pH

Endocytosiso Phagocytosis

Ingestion of large moleculeso Macropinocytosis

Membrane grows around Fluid uptake

o Clathrin-coated vesicles Forms a membrane enclosed vesicle

o Non-coated vesicleo Caveolae

Smaller vesicle (50-80 nm) Lysosomes

o Four processes Phagocytosis Autophagy Endomembrane transport

Receptor mediated endocytosis Early endosome matures to late endosome

o Lysosomal storage diseases Substances accumulate inside of the cell

Endomembraneo ER, golgi, and ribosomes work together to produce proteinso Proteins synthesized in the rough ERo Move to golgi for processingo Travel to the cell membrane or other destinations

ER signal hypothesiso Life begins in cytoplasmic ribosomeso Signal sequence is synthesized by ribosomes

About 20 AAo SRP binds to signal, pauses translation, and brings it to the rough ERo SRP receptor allows translation to continueo SRP signal is removed

Trans golgi networko Form a vesicleo Get a mature lysosomeo Maturation process

Cytoskeleton

Composed of protein fibers Gives the cell shape and structural stability Aids cell movement and transport of materials within the cell Organizes all of the organelles and other cell structures into a cohesive whole Disrupt the cytoskeleton?

o Entire intermembrane system is destroyed 3 types of cytoskeletal elements

o Actin Along the outside (cortex) of the cell

o Intermediate filamentso Microtubules

Organize the organelles Microtubules

o Made of alpha and beta tubulin dimerso Have polarityo Dynamic (can grow and shrink)o Usually grow from the plus end of the cello Function

Stability Movement Structural framework for organelles and a track for intracellular transport

o Originate from Microtubule Organizing centers (MTOC) Major one is the centriole in animals

o Taxol Molecule that stabilizes microtubules to study them

o Colchicine Depolymerized MT

o Motor proteins that use ATPase enzymes to move Kinesin

Towards the + end Dynein

Towards the – endo Form cilia and flagella

Actino Microfilamentso Grouped in bundleso Found just inside the cell membrane in the cortexo Two strands of actin are twisted together for strengtho Play a role in muscle movement

Intermediate filamentso Provide structural support for the cello Not involved in movemento Connected cell to cell or cell to ECM to help anchor the cell

Cell Adhesion

Cells are physically connected to the ECM ECM

o Everything outside of the cello Peripheral proteins are usually anchored by actin filaments on the inside of the cell

Integrin (in the PM)o Attaches to fibronectin in the ECM which binds to collageno A way for the cell to anchor

Collageno Part of the ECM (main component)o Most abundant protein in the body

Fibronectino Dimer

Linked by disulfide bonds

o Binds to integrins Tight junctions

o Forms between PM of adjacent animal cells to form barriers that allow adheringo Membranes are pinched together and have membrane proteins to form a tight junctiono Usually found in tissueso Tight water line

Desmosomeso Adhering of cell’s intermediate filamentso If damaged, can lead to blistering diseaseso Proteins in between=cadherins

Gap junctionso Allow cells to communicateo Allow small molecules to pass througho Can be gated or ungated

Selective adhesiono Cell-cell connections are species and tissue specific

Signal transduction

Every cell interacts with its environmento Relay signals from outside in

Receptorso Found in the plasma membrane for signals that cannot pass through the PMo Found in the cytosol for signals that can pass through the PM (lipids/steroids)

Receptor usually becomes a transcription factor and goes into the nucleus Binding creates a shape change in the receptor, activating it Target cells are very specific

o Long distance signaling uses the bloodo Short distance signaling is local growth factors

Transmembrane receptorso Shape change leads to

Activation of cytosolic protein that trigger the production of intracellular messenger molecules

Enzyme linked receptors (Tyrosine kinase) that create a phosphorylation cascade that activates a series of proteins in cell

G-proteinso Activated by GTPo Steps

Hormone binds to receptor Change activates the G protein G protein binds GTP

G protein activates downstream enzymes which signal or generate second messengers

cAMP, Ca, cGMP, DAG, IP3 second messenger cascade amplifies the signal and increases the reaction

o deactivation G proteins hydrolyze GTP to GDP 2nd messengers degraded or pumped back into storage (Ca ion) Cascades are turned off by phosphatases

enzyme linked receptorso directly phosphorylize kinase by the receptor instead of using a 2nd messengero TK form a dimer when activatedo Steps

Signal binds Triggers a receptor cross phosphorylation Causes Ras to bind to GTP and receptor through bridge proteins Ras triggers a phosphorylation cascade

Phosphorylation by a kinase Dephosphorylation by a phosphatase

Cell cycle

Basic detail (see cell bio notes for a complete chapter on cell cycle) Five steps of mitosis

o Prophase Chromosomes condense and the spindle begins to form

o Prometaphase Nuclear envelope breaks down Kinetochore microtubules connect

o Metaphase Chromosomes complete migration to middle of the cell

o Anaphase Sister chromatids separate Pulled to opposite sides of the cell

o Telophase Nuclear envelope reforms and spindle disintegrates

Cell division than begins (cytokinesis)o Myosin and actin interaction

Mitosis varies somewhat in organismso Not always by the 5 steps

Experiments tell us that the chromosome is pulled away from the spindle from the + end to the pole

o And that the + end disassembles

Cytokinesiso Once segregated, need to divide the cytoplasmo Plants

Cell plate needs to form so that a new cell wall can formo Animal cells

Existing membrane is pinched by actin/myosin contratction Regulation of the cell cycle

o Signals are given to the cell to start the cycleo 2 types of proteins in cell cycles

Ones that are fundamentally required to proceed Ones that are only important if something goes wrong and needs to be

correctedo Mitosis promoting factor (MPF)

Induces mitosis Cyclin/cdk complex

Cyclin cycles Cdk remains constant

Checkpointso G1o So G2o Mo Don’t complete the cell cycle if DNA or chromosomes are damaged

Mitogenso Proproliferative growth factorso Influence a cell’s decision to enter S phaseo The RB protein stops s phase entry until it is deactivated by the mitogen

P53o Guardian of the genomeo Acts as a DNA damage sensoro Can halt the cell cycle so DNA can be repairedo Detects mutations

50% of human cancers have a p53 mutation Failing to control the cell cycle

o Cancero unicellular

Defect in nutrient sensing allows the cell to be outcompeted for nutrients Defect in starvation censing leads cells to grow in the absence of sufficient

nutrientso Multicellular

Defect in sensing signals for growth leads to a failure to develop

Defect in signals to arrest growth would lead to cancer, which is not a single disease but a collection of different defects

Meiosis

Sexular reproduction must have some evolutionary advantageo Puryifying selection

Sexual derived progeny are less likely to have deleterious alleles They get an evolutionary advantage

o Changing environment hypothesis Sexual reproduction gives greater diversity, and greater chance for adaptation Asexual reproduction is fine when the environment remains stable

Karyotype-complete set of chromosomes in a cell Diploid-contains two non-identical copes of each chromosome One pair of homologous chromosomes

o Same set of genes in the same ordero Contains different alleles

Humanso 22 autosome pairso 1 pair of sex chromosomeso 46 total

Meiosis halves the number of chromosomes to create haploid cells Only occurs to create germ cells (egg/sperm) Tetrad of chromosomes forms Meiosis I

o Reduces the number of chromosomeso Daughter cells are haploid but with replicated DNA

Meiosis IIo Stay haploido Now have 1 copy of each chromosomeo Not replicated

From one cell, you get 4 haploid gametes Most important step in meiosis is crossing over that occurs in late prophase I

o Genetic recombinationo Produces new combinations of alleles

Heredity

Mendel’s model peaso Self pollinationo Cross pollination

Tests revealed

o Mathematical rules of heredityo Phenomenon of dominant and recessive traits

The test results contradicted the blending-inheritance hypothesiso Which was just a 50/50 blend of 2 parents

Control experiments needed for conclusionso He worked with pure line strains

Polymorphico 2 or more alleles

Particulate inheritanceo Trait maintains its integrity through generationso Easy to assess

Single phenotype (in this case) is controlled by a single geneo Dominanto Recessive

Refers to the relationship between alleles Dominant is expressed in the heterozygote

Wildtypeo Defines a reference alleleo Mutant is a change in function from the wild type

Punnet square is based on Mendel’s observations

Test cross determines what the unknown allele is

o Uses ratios of the offspring to tell

o Traits segregate independently because the segregation of homologous chromosomes during

anaphase I is random The genotype in a daughter cell depends on lining up and segregation during anaphase I

o Segregate based on how they line up Mendel’s genes were all on different chromosomes

o Segregated independentlyo If they had been on the same chromosomes they would not have segregated

independently Sex chromosomes

o Genes present on sex chromosomes are sex linked

o Human y chromosome does not have many genes on it Males only have one x and one y, so they only display the defective phenotype

o Have no second copy to cover The closer 2 genes are located on a single chromosome the more linked they are

o Crossing over is rare between themo Linked genes segregate together EXCEPT when crossing over occurs

Incomplete dominanceo If a genotype is heterozygous, it will appear as a mix of the dominant and recessiveo Close to blending inheritance but the second generation ratios were not correct

Codominanceo Two alleles are phenotypically expressed in equal measureso Example is blood type AB

Multigenic traito Interactions between different genes affect phenotype

Pedigree analysiso Very useful to helping figure out what disease it is and how it is passed ono Autosomal-both males and females affectedo Recessive-affected children without affected parentso Dominant-one parent affected and all children affectedo X linked

Only sons get the disease Daughters are carriers

o Y linked Male to male (since cannot get a Y from anywhere else)

DNA and the gene

Chromosomes are 60% protein (histones) and 40% DNA Hershey-Chase Experiment showed that DNA is the hereditary material

o Marked phosphorus (DNA) and sulfur (proteins) and saw the presence of tagged phosphorus in the offspring

Transforming principle (Griffith experiment) confirmed DNA is the genetic materialo Something from the dead virolent can transform nonvirolent into virolent

o DNA structure

o Nucleotide 3 components

Sugar (deoxyribose) (5 carbons) Phosphate group Nitrogen base

o Sugar Deoxyribose in DNA Ribose in RNA

o Phosphate group is added to the 5’ end o Sugar base bound to the 1’ endo Bases

Pyrimidines-single ring structure C, U, T

Purines-2 ring structures Guanine and Adenosine

Condensation reaction is a phosphodiester linkageo A-To G-C

Bond between an O and a phosphate group 2 strands are antiparallel The DNA ladder twists to form a right handed helix to optimize the interactions between base

pairs The purines need to bind to the pyrimidines to maintain a constant diameter RNA is a more versatile molecule because it is single stranded so it can form loops and twist Replication

o One strand serves as a blueprint for a second strando Add to the 3’ end

Can only go in the 5’ to 3’ directiono Primase

Creates a RNA primer so DNA polymerase can attach and begin replicationo DNA polymerase has a proofread functiono Replication is semiconservative

Not conservative or dispersive Found experimentally with Nitrogen markers

Half and half in first gen, half and half and some completely new in second gen

o Replication begins at replication bubbles It expands in both directions Prokaryotes have only 1 origin Eukaryotes have multiple replication bubbles Leading strand is in the 5’ to 3’ direction Lagging strand is 3’ to 5’

Requires many more primers Okazaki fragments

o Joined by ligase by covalent bondso DNA polym I

Removes the RNA primero DNA polym II

Extends the leading stran/okazaki fragmento Problem when the end of the chromosome is reached

There is a small end that cannot be replicated Telomeres

At the end of the chromosome to prevent the real DNA from being eaten away

Telomerase A protein that can synthesize the repeats Allows chromosomes to replicate without losing valuable genes Not found in many cells

Shortening occurs on the leading strands in addition to the lagging strand

Changes to DNAo Replication errorso Spontaneous damageo Chemical/radioactive mutagenso Transpositiono Viral transduction

DNA repairo Nucleotide excision repairo Base excision repairo Mismatch repairo Double stranded break repair

Excision repairo Usually thymine dimers

How Genes Work

Genes code for proteins DNA to RNA = transcription by a molecule called RNA polymerase RNA to Protein = translation by ribosomes 3 bases in mRNA codes for a protein

One gene one enzyme hypothesiso Proposed that each gene contains the info needed to make an enzyme

Beadle and Tatum used bread mold to test the hypothesiso They damaged DNA to create mutants that had new nutritional requirements

Srb and Horowitzo Tested the one gene one enzyme theory using the Arganine pathway

Central Dogma of molecular biologyo DNA is the information storage materialo Sequence of DNA calls for Amino Acidso The base sequence must first go through RNA before it becomes a protein

mRNA carries info from the DNA to the site of protein synthesis mutations can occur Transcription identifies the template DNA strand to limit the choices to the three possible

peptides Translation can identify which frame to read A single point mutation in the DNA can have huge effects on the protein

o Ex: sickle cell anemia Exceptions to the central dogma revolve around functional mRNA molecules that are not

translated and perform other functions in the cell Some viral genomes also contain reverse transcriptase, which takes RNA and inserts it into the

DNA of the host cello Mostly retroviruses such as HIV

20 total AAo 64 possibilities based on the 3 codonso Repeats (redundant)

Nirenberg and Leder o Cracked the codons by creating RNA moleculeso Found that the start codon is AUGo There are 3 stop codons

UGA, UAA, UAGo Many organisms can incorporate a 21st AA

Selenocyteineo And some Archaean bacteria have a 22nd

Pyrrolysine Code

o Redundanto Unambiguous (only a few exceptions)o Nearly universalo Conservative

Using the Codeo Predict codons and AA sequence encoded by a particular DNA sequence

o Approximate the mRNA and DNA sequence that codes for a particular sequence of AAo Engineer specific changes to a protein in order to determine the consequences of such a

change Mutations

o Can be point (one change) Missense-one AA replaced Silent-no change to the AA Nonsense-premature stop codon Frameshift-shift the whole frame + or – 1

o Or can be chromosome level Polyploidy-increased number of chromosomes Aneuploidy-addition or deletion of chromosomes Inversion-within the chromosome it breaks off and reattaches somewhere else Translocation-moves to a different location

o Mutations can be: Beneficial to increase fitness Neutral (no effect) Or deleterious and decrease the fitness of the organism

Transcriptiono DNA to RNAo Gene expression describes the production of a functional product (RNA or protein)

Different processes regulate gene activityo RNA

Synthesis Processing Degredation

o Protein Synthesis Modification Localization Degredation

Transcription is the synthesis of a single stranded RNA complementary to a DNA template strand H bond between bases Phosphodiester bonds between sugar and phosphate backbone RNA is synthesized in an antiparallel complement to the template strand RNA bonds

o Phosphodiester bond between OH on 3’ carbon and the phosphate group on the next nucleotide

ORFo Open reading frameo Stretch of sequence starting with a start codon and ends with a stop codon

Prokaryoteso 1 RNA polymerase

Eukaryoteo 3 types of RNA polymerases

I-most rRNA genes II-protein coding genes, miRNA, plus some of the small RNAs III-tRNA and some other types of RNA

Prokaryoteso Promotor=start signal

Where RNA polymerase starts the process of transcriptiono Attaches to a TATA box

Found on the nontemplate strando A preinitiation complex assembles

RNA polym II CTD Other factors (transcription factors)

o Required for transcription to initiate Prokaryotic Termination

o The presence of the 2’ OH in RNA allows greater flexibility, this allows greater flexibility to generate hairpin that triggers termination

o A hairpin loop forces a termination that yanks the RNA out of RNA polymerase Eukaryotic termination

o Processedo Factors recognize that a sequence is telling them to stopo Splicing activity needs to occur

Exons Coding (expressed)

Introns Noncoding

Some DNA does not have complementary RNA (introns) Introns are removed by splicing and the exons are spliced together to create

one RNA strand Cap and tail are still included Spliced by a spliceosome

Small nuclear ribonucleoprotein (snRNP) snRNP ID’s the ends of introns and binds to them

o 3 other things happen to RNA 5’ end is capped for protection Splicing of introns out and exons together Polyadenylation –add a poly A tract to the 3’ end

Eukaryotes tend to only code for one protein

o Some exceptionso Alternate patters of splicing can produce multiple forms of a protein from one transcript

Enhancerso Cis acting that bind to trans-acting factors in Eukaryotic cells

Prokaryotes translated right as its being transcribed Southern blots=DNA Northern blots=RNA Translational

o Synthesis of proteins from an RNA templateo Take the RNA molecule outside of the nucleus to the ribosomes in the cytoplasmo Ribosome needs to recognize mRNA as a substrate and reading it

tRNAo 3’ end is the amino acid acceptor arm and has an internal anticodon base pairs with

codons in the mRNAo Binds to and brings a tRNA to the ribosomeo 3 loops for a tRNA

2nd loop contains the anticodono Net result-AA is selected by its codon

Creates a high energy bond between 3’ and AA tRNA becomes charged when bound to an AA

o some tRNA can wobble means that there can be a mismatch in the 3rd position of codon-anticodon pair explains why there are fewer tRNA than codons

Inosineo 5th Nucleic Acido Modified derivative of Guanosine

Ribosomeso Eukaryotic

49 ribosomal proteins, 3 different rRNA molecules make up the large (60s) ribosomal subunits

33 ribosomal proteins, 1 different rRNA creates the small (40s) subunito Creation of ribosomal subunits takes place in the nucleoluso Assemble into a full ribosome in the cytoplasm when bound to an mRNA

rRNAo don’t code for proteinso transcribes from geneso recognize and bind RNAs and proteinso catalyze the peptide bond between AAo 80% of RNA is ribosomal

Peptide bond formation lengthens AA chain in the 5’ to 3’ direction E, P, and A sites in the ribosome

o A is for aminoacyl site where the tRNA enterso P is for the peptide site where the bond formso E is for the exit site

Peptide transferase reactiono Accomplished by the transfer of AA from tRNA in P site to the A site

Eukaryoteso The 5’ cap binds to the 3’ tail and creates a loop, this allows the binding of the small

ribosomal subunit to the 5’ end Shine dalgarno

o Ribosome binding site in bacterial RNA (mRNA)o Specifically tells what reading frame (since there are multiple ones

Terminationo Release factor binds to stop codon

No charged tRNA Special release factor

o Will cause a release of polypeptideo Large subunit is first to leave

In bacteria, transcription and translation are tightly coupled allowing for a mode of transcription regulation based on availability of certain tRNA

Can transcribe and translate at the same time Eukaryotes separate transcription and translation in time and space Molecular chaperones

o Help proteins find their correctly folded shapeo Bind to hydrophobic surfaceso HSP70 (heat shock protein)

Helps the protein refold after it denatures

Gene Expression Prokaryotes

Gene expression can be regulated at any stage from mRNA synthesis to protein activity Types of regulation

o Transcriptional Saves the cell the most energy Don’t transcribe all the genes Only the ones that need to be used are transcribed

o Translational control Least common Used to control levels of some ribosomal proteins Controls whether or not ribosomes will translate mRNA

Charge mRNA life span Change translation rate

o Post translational

Speed of response is of the greatest importance Energy is expensive but creates a fast response Proteins are activated/inhibited by chemical modifications Proteins get made in the cell but the activation signal usually comes from

outside There are many control points of gene activity Transcription controlled

o RNA abundance is alteredo Protein abundance is alteredo No change in other aspects

Translationalo RNA abundance is not changedo Protein abundance is altered (does not translate)o Other aspects unchanged

Post translationalo RNA no change (already a protein)o Protein abundance changes

If RNA levels change…o Can figure out if it is reduced transcription or increased degredationo To differentiate, we block transcription from taking place

Learn about how fast RNA is being degraded Transcriptional regulation: Protein-DNA interactions

o A transcription factor is a DNA binding protein that influenced the level of transcription of a gene, either positively or negatively

o They bind to promotorso Specific minor/major grooves that recognize specific binding sites on DNA

Consequences of protein binding to DNAo Hinder RNA polymerase from recognizing the promotor region

If a repressor is bound to an operator region no transcription occurso An activator may bind near RNA polymerase promotor site

This draws the RNA polymerase to the promotor region Repressors or activators are referred to as trans acting factors Site where they bind is a cis acting element CIs mutations

o 2 mutations on the same DNA molecule Trans mutation

o 2 mutations on different DNA molecules Use cis/trans test to determine whether or not mutations are present on the same gene or not

o

Cis dominant mutations only affect the function of the DNA molecule on which they are locatedo Haploid-automatico Diploid-have another to cover, can be recessiveo On the same strand

Phenotype is wild type and the function is limited by the genes acitivity that is mutated

Transo On different strandso Elevates phenotypeo Mutation on upper chromosome appears to be dominant

Constitutive mutationso Always ono Express the gene even when the promotor is not being targeted

Catabolite repressiono Form of feedback inhibition, the product of a pathway inhibits upstream components of

the pathway Back to translational control

o N-end rule The amino end of the AA determines the half life of the protein

Synthsiso mRNA binds to small subunit via the ribosome binding siteo F-Met tRNA binds (initiator codon)o Large subunit binds

Secondary structure of the mRNA can hide the Shine Dalgarno (SD) sequence so that the small ribosomal subunit does not bind, and translation is not initiated

o Basically the RNA itself is regulated instead of the ribosomes The SD sequence also can form a stem loop to block access

o Called riboswitches Thermosensative Could respond to proteins too

Feedback regulation tells the cell to stop making a certain product if there is enough of ito Saves the cell energy

Also a regulation to the presence of ribosomal subunitso Need an equal amount of the small and large subunits

Post translationalo Protein phosphorylationo phospho-relay (cascade)o Protein methylation

Detect post translational modificationso Western blots can detect a mobility shifto If a mobility shift is observed one can use an enzyme to remove the modification and

see if the mobility ceaseso Mutating the predicted site of modification should alter the mobilityo Tag the proteins with antibodieso Analyze using mass spec

Gene expression Eukaryotes

Variety of mechanisms to control gene activityo Chromatin (unique)

Protein/DNA complexo Nucleus

Chromatin remodeling Euchromatin-active Heterochromatin-inactive

Transcription – pre mRNA RNA processing

Cap and poly-A tailo Cytoplasm

mRNA stability translation post translational modifications

every step from heterochromatin to active protein can be modified chromatin

o histone protein and DNA

o 8 subunits of histones with DNA wrapped = nucleosomeo Organized DNAo Nucleases can take DNA off for translation

Chromosome is interspersed with hetero/eu chromatin Condensed uncondensed

o HAT (Histone acetyl transferase) adds acetyl groups to decondense chromatin Uncondensed condensed

o HDACs (Histone deacetylases) remove acetyl groups to condense chromatin Enhancers in a gene are binding sites for transcriptional activators that are not near the

promotero Can be found upstream or downstream

Core promoter binds to general transcription factors Promotor proximal elements are binding sites for transcriptional activator or repressors that are

near the core promotor If an enhancer is translocated to a different place, it can enhance a different gene Transcriptional control allows cells to respond to environmental changes Epigenetic inheritance

o Inherit the same patterns of histone modificationso Have same hetero/euchromatin

X chromosome inactivation in women creates 1 barr body Transcription initiation depends on an initiation complex

o The TF binds to DNAo Chromatin loosenso TF bind to enhancers and promoter proximal elementso Basal transcriptional complex begins transcription

DNA is dissociated from the nucleosomes by RNA polymerase Post translational control

o Once an mRNA is made, a series of events occur if the final product is going to affect the cell

o 4 major control points Alternative splicing of exons

Takes place in the nucleus Benefit is that different forms lead to different domains that can lead to

different functions Alter the rate of translation initiation Alter mRNA stability Post translational modifications to alter protein abundance, localization, or

interactions RNA stability and RNA interference

o In the cytoplasm, there are mechanisms to control gene expressiono Regulate the amount of DNA translated

Block access to mRNA by ribosomes Alter stability of RNA

Highly variableo Some degrade rapidly, others are very stable

o Life span of mRNA is controlled by RNA interference Specific mRNAs are targeted by microRNA (miRNA) Binds to parts of the RISC protein complex Degrade the mRNA or prevent translation

o 20-23% of all animal and plant genes are regulated by miRNA mi vs si RNA

o miRNA dsRNA from an endogenous gene no perfect base pairing mRNA level is unchanged there is a reduction in protein

o siRNA dsRNA from a foreign source perfect base pairing leads to RNA being broken down mRNA levels reduced protein levels reduced

translation is controlledo miRNA blocks translationo mechanisms can control the time and rate of translationo translation can be slowed or stopped by phosphorylation of ribosomal proteins

post translationalo speed of response is fasto cells can respond to new conditions rapidly by activating or deactivating proteinso types

chaperone protein enzymes can modify proteins by adding groups Proteins can be activated/deactivated Targeted protein destruction