bio

Termination (prok)

- find a terminator sequence of a G-C base pair followed by a strang of U= weak -physically pulls transcript out of the polymerase, allowing the RNA product to release and the helix to form -stem loop reduces the length of the hybrid

Rifampicin and actinomycin in terms of transcription

- inhibit transcriptoin through blocking the exit of RNA or intercalate between the double helix in euks and proks= cancer drug

rRNA and tRNA modified post transcription

- mRNA is not modified post transcription because it is used right away -many tRNA modifications lack CCA sequence at 3' end of strand

Identify the factors that make ATP an energy rich molecule and explain how ATP can power reactions that would otherwise not take place

-ATP and ADP contain unstable bonds- phosphoanhydride bonds -ATP/ADP hydrolysis is energetically favorable. -Use energy that is released to host cellular functions -Resonance stabilization -Electrostatic repulsion -Increase in entropy -stabilization by hydration

Explain how protein misfolding and affregation are associated with diseases

-Amyloidoses are diseases that result from the formation of protein aggregates (Alzheimer) -Normal protein conformations can exist in forms rich in beta sheet, which are prone to aggregate -Prion diseaes (mad cow)- good proteins gone bad

Define introns, exons and explain what is meant by alternative splicing and exon shuffling

-Introns are cleaved out, exons remains -mRNA precursor is often spliced, removing introns that do not encode protein sequences. Often, the same pre-mRNA is spliced differently in different cell types or at different stages. (alternative splicing allows the generation of multiple proteins from a single gene,contribute to temporal control of gene expression, produce nonfunctional mRNAs, be tissue specific) -exons contain domains= each forms a fully functional protein polypeptide chain -exon shuffling= using recombination, the exons can recombine to have mRNA containing each domain. This can create new coding sequence

Describe how tRNA is processed

-RNA polymerase 3 -Promoters are type 1: 5S rRNA= c block and Type 2: tRNA= b block -all within transcript -snoRNA is made= modify rRNA as synthesized -snRNA -miRNA -processed post transcriptoin= RNA polymerase 3 catalyzes precursors to tRNA - RNase P cleaves off leader sequence -CCA-adding enzyme adds nucleotides to the 3' end -many precursers have an intron removed by endonuclease and then rejoined by ligase

Given a consensus sequence, determine if a promoter would be considered strong or weak

-close to the core promoter sequence -10 or -35 -need both reigons for initiation of transcription -makes contact with 50 bp, DNA melting importnat, 10 nucleotides required

Differentiate between three eukaryotic RNA polymearses and their promoters

-differ in DNA substrate, location, and sensitivity to toxin called alpha amanitin (found in deathcap mushroom= inhibits RNA polymearse 2) -all RNA polymearses are not inhibited by rifampicin (as opposed to prokaryotic e. coli) -2 (mRNA)>3(5SRNA)>1(18SRNA) for alpha amanitin -RNA polymerase 1 found in nucleolus, found in the ribosome making factory= rRNA transcriber

DNA polymearse/ RNA polymearse differences in structure, function, mechanism of action

-do not need RNA primer= de novo -RNA polymearse backtracks through use of water molecule, will have a chance to re base pair two bases -no exonucleases - error rate in RNA is much higher because it is unstable and short lived

Compare and contrast the self-splicing introns with that of the spliceosome-catalyzed splicing of nuclear mRNA

-group one and group two are self splicing= they require no additional proteins or ATP -in self splicing group 1, the 3'OH of free guanosine is a nucleophile, attacking the bond between the U and A at the end of the intron. group two uses 2'OH (two transesterification reactions) -splicosomal introns are spliced by spliceosomes= most common -snRNA are catalysts in splicosomal intorns -

Sigma factor in prok transcription initaiton

-holoenzyme= all 5 RNA polymerase subunits -sigma identifys promoters -once bound, DNA will unwind to expose the template starting transcription -binds to RNA polymerase as a subunit

Define and explain the role of promoters in trancription initation

-in prokaryotes there is a core promoter reigon that is recognzied by the sigma subunit on polymerase -strong promoters will have lots of transcripts and bind tightly -promoters will be negative, upstream -more similar to the most frequent will be the stronger

Explain how a variety of antibiotics and toxins can inhibit protein synthesis+examples

-inhibit protein syntheiss in prokaryotes -stretomycin= binding of formylated methionine tetracyclines= block a site on ribosome chloramphenicol= blocks peptidyl transfer -some inhibit both= exhibit property in both= puromycin= similar structure to aminoacyl trna so binds to a site of ribosome, forms bond with growing peptide (premature chain termination) -toxins = diptheria toxin blocks translocation - covalently attaches to eEF2= no longer function properly -ricin inhibits 28S rRNA= cleave an adenine off of 28S ribosomal rna, cant bind elongation factor

List common post translational modifications and describe purposes

-polypeptide binds cofactors -many require covalent modifacation -glycoslyation -enzymatic removal or a formyl group from first residue -removal of signal sequences -attaching carbohydrates -acetylation of n-terminal residue -phosphorlyation of ser -adding lipids to different proteins to help anchor into membranes -prosthetic group- heme and hemoglobin

basal (general) transcription factors

-protein-protein contacts -bind to promoters to regulate gene expression -bind to TATA

Elongation (prok)

-ribonucleoside triphosphate base pairs with a nucleotide on the DNA template - RNA polymerase can backtrack - happens in transcription bubble- hybrid between DNA and RNA -happens until a termination sequence

Describe the roles of various snRPs in the spliceosome splicing process

-ribozyme -Catalyzing reactions -U1 binds at splice site, U2 binds at branch site -U4,5,6 tri snrp completes the formation of the spliceosome -ATP required, some parts attached to CTD

How proteins are turned over in a cell

-several hundred complexes that regonize the degradation signal on target proteins that indentify the target -degradation signal has free lysine, will reconzise and ubiquitinize -Protein degradation is inevitable= hemoglobin is long lived while defective are short lived -specific sequences on the proteins determine if they will be degraded sooner rather than later (degrons)

Proteins involved in Eukaryotic mRNA transcription

-transcription factors -TF2 TF2A TF2B -highly conserved -RNA pol 2 is a large complex of 12 subunits -CTD have many repeats -TF2H is a helicase and a kinase allowing transition from initation to elongation (also nucleotide-excision repair NER)

Why is sigma factor considered catalytic and why bacteria uses various sigma factors instead of one

-will bind with RNA polymerase and once it gets past it will fall off and bind to another RNA polymerase

Describe the 5 principles that underlie energy flow

1) Metabolic pathways= build things up and break things down 2) ATP is energy currency of life 3) ATP can be formed by oxidation of carbon fuels 4)Can be broken down into classes 5) Metabolic pathways are highly regulated

Describe six steps in which eukaryotic genes can be regulated, and give one example of each.

1) Transcriptional control 2) Rna processing control= alternative splicing, alternative poly a site, RNA editing 3) RNA transprt and localization control=mRNA export, mRNA targeting (ER membrane for cotranslation processing) 4) Translation control 5) mRNA degradation control= miRNA= can silence 60%, mRNA degradation pathways 6) Protein activity control= post-translational modifications

Describe the three stages of energy extraction from foodstuffs

1) digestion 2) small molecules degraded 3) used in body

Define and provide an example of 1)operon 2)operator 3)inducer 4)repressor 5)activator

1)operon= a cluster of genes under one regulatory sequences. Operon contains control sites and regulator genes. Will encode repressor. 3) inducer= bind the repressor, preventing it from binding the operator and allow transcription 4)repressor= halt transcription from binding to the operator lac repressor= protein that binds to the regulator site (operator). Nucleotide sequence shows an inverted repeat, twofold rotational symmetry. DNA binding domain from lac repressor binds to operator DNA by inserting an alpha helix into the major groove of operator DNA

Define the wobble hypothesis and explain how it is posible to have one anticodon recognize multiple codons

1. Codons that differ in either of the first two nucleotides must be regognized by different trnas 2. The third base of the anticodon determines the degree of wobble. If the first base is inosine, the anticodon can recognize three codons (first two positions of the codon have checking to ensure it is correct, rRNA does not evaluate correctness of the third pairing)

List and describe the general characteristics of the standard genetic code

3 nucleotides= codon nonoverlapping= each three encodes a separate amino acid -organisms use the same genetic code -

Activators, barriers, insulators

Activaros have separate DNA-binding and activating functions. They enhance transcription by binding directly to general basal transcription factors, to a mediator, or co-activators, or recruit other histone modifying enzymes. INSULATORS ensure that activators act appropriately, BARRIERS prevent spread of heterochromatin which would silence gene. Activator binds to enhancer which can do its job.

Beta amyloids

Alpha helical turns to beta amyloid strucutre, very stable, turns all the other proteins bad because they are converted into the bad structure

Compare and contrast the structures and functions of the prokaryotic and eukaryotic ribosomes

Bacteria- ribosome has two subunits, large and small. 2/3 of the subunits is rRNA eukaryotic- similar shape, just bigger = different rrna, same function Polysomes in prokaryotes and eukaryotes= multiple ribosomes translating the same rNA

Explain how the lambda repressor/ cro protein regulatory circuit functions to control lytic and lysogenic stages of the lambda bacteriophage

Bacteriophage has two alternative infection modes. Circular DNA can go into lytic phase or lysogenic phase. Lytic would produce all genes, lysogenic is incorported into the genome. Lambda repressor keeps the prophage silent, not produce any genes besides lambda repressor, when it switches to lytic phase, it gets eaten up and all other genes are expressed. Lytic= all viral protiens expressed, lysogenic phase ONLY has lamda repressor expressed. CRO turned on will turn off lambda repressor and turn off everyone else. Binds cooperatively = n terminal domain has a helix turn helix motif, C terminal has a tetramerization and dimerization region.

Amphibolic

Can do both anabolism and catabolism (Krebs cycle)

Catabolism

Combust carbon fuels to synthesize ATP- burn

Metabolism

Composed of many coupled, interconnecting reactions

mediators

Connect transcription factor to machinery at polymerase= facilitate communication between the CTD of polymerase, provides assembly

Explain the difference between constitutive expression and regulated expression

Consistituve expression- housekeeping gene, always expressed, simple gene strucutre, -10, -35 signal sequence Regulated genes= inducible is turned on when they need them, repressible is able to be turned off when you dont need them -regulated gene strucutre is more complex -Regulate transcription --sigma factors expressed in differnt conditions --other proteins transcription factors acivating or repressing transcriptoin

Descrbie how attenuation works

Control gene expression in proks. From a premature terminator in the mRNA before the structural genes. This is formed prior to transcriptoin

Explain how proteins fold once synthesized

Cooperative process Amino acid sequence determines 3D structure Most proteins show a sharp transition from folded to unfolded with increasing concentrations of denaturants. Progressive stabilization or cumulative selection= monkey typing= partly correct folding intermediates are retained because they are slightly more stable than unfolded reigons.

Define combinatorial control, and provide example.

Dimerization of the gene regulatory proteins- more than one transcription factor required for full activity. The two coming together gives diversity of binding specificity, gives you a wide variety of different examples.

Outline how proteins can be sorted and targeted to different organelles and the plasma membrane

Eukaroytes can direct proteins to distinct organelles Secretory pathway= ER co-translational targeting. Ribosomes that will end up on the rough ER. Protein getting translated has a specific sequence and will continue synthesis inside lumen. Sequence starts with lysine or argenine, then a long hydrophobic residue chain. N-terminus will have a signal sequence to target the protein directly to where they need to go

Cis-acting elements

Eukaryotic promoters= same molecule- DNA segment These regulate gene expression on the same molecule

CRISPR/Cas system

Gene function. Clustered regulary interspaced short palindromic repeats= use DNA instead of RNA= came from DNA viruses infecting the bacterial cells. Repeat sequences are present

RNA interference- genetic knockdown

Gene function. Silences gene expression. Use double stranded RNA and dicer will created siRNA= bound to mRNA. These are loaded into an assembly of several proteins called RNA-inducing silencing complexes. These will cleave mRNA down the middle and silence mRNA before the ribosomes can translte them. Similar to microRNAs= encoded by genome and transcribed by RNA polymearse 2. Exported into cytosol -can shut down genes artifically= bind to rna to silence translation= need double stranded rna

Genetic knockouts

Gene function= remove or replace gene with genetic marker to take it out. Mutated gene, create on plasmid, use homologous recombination to create mutation in targeted gene. Then we can see what the targeted gene function is based off the phenotype. Knockout mice will have mutated gene and both allelesl of their gene. (See what function of 123 genes are by replacing them with a selectible marker)

Compare and contrast DNA-binding motifs found in gene regulatory proteins.

Gene regulatory proteins Bind DNA and help regulate transcription initation. Most gene regulatory proteins bind as dimers in the major groove. A wide diversity of motifs has a high diversity of DNA. This adds to the specificity of their binding. DNA binding motifs have critical interactions in the minor groove of DNA as well.

Explain the regulation involved in Trp operon

If trp is high, the attenuator will form. If trp is low, the alternative secondary strucutre will form, allowing transcriptoin. The tryptophan attenuator only forms when Trp is plentiful, and thus is availible for attachment onto the tRNA. When trp is low, the ribosome pauses at the trp codons in the sequence, and formation of the paried structure prevents attenuation.

Describe how riboswitches work

Inhibit transcription at termination level. Riboswitches exploid rho independent terminator- low levels of guanine high levels of guanine will bind rna as transcribed and make premature termination, making it not transcribed.

Define and give an example of quorum sensing

Interaction among bacterial cells , bacteria synthesize luciferase. an autoindcer that uptake other cells which simulate the luciferase gene. Cells can "talk to each other" in biofilms, large communities of bacteria that are resistant to the host immune response as well as to many antibioticsc.

Ferritin and transferrin receptor are controlled at transitional level.

Iron is a key componnet of many proteins, including cytochromes. Transferrin is a blood protein that transports iron, which binds to a receptor. Ferritin is an iron storage protein in the cell. Ferritin mRNA coding for the storage contains a stem-loop structure in its 5' untranslated reigoin called the iron response element. Transferrin receptor binds transferrin , when iron is in high amounts, iron binds to IRE-BP causing it to dissociate from the transferrin receptor = presence or absence of iron regulates metabolism genes. No iron= only receptor is made (irp bound to both ire), ribosomes translate protein and protect ires Iron= iron binds to irp, exposing stem loop strucutre, allowing ferritin to be made.

Explain why most eukaryotic genes are regulated at the level of transcripion initiation over any other step.

It is at the begining so you can limit have a buildup of things you do not need.

Describe how allolactose and cAMP combine to regulate the lac operon

Lac operon regulates the lactose levels in e. coli. When lactose levels are low, transcription is repressed because the lac repressor is bound to dna. When lactose levels are high, transcription is no longer repressed and turned on. cAMP is produced when the glucose levels are low. cAMP rises in levels, signaling that there are low levels of glucose. CAP binds to cAMP, helping the polymerase to bind tightly and activate transcriptoin. FOR ACTIVATED TRANSCRIPTION OF THE OPERON, LACTOSE IS HIGH AND GLUCOSE IS LOW. Lac operon are maximally expressed hen allolactose relieves inhibition by the repressor AND CAP-cAMP stimulates RNA polymerase binding. Basal transcriptoin is when low expression when lactose is high glucose low.

RNA polymerase 2

Many promoters recognized y RNA polymerase 2 contain regulatory elements several kbp upstream of the initiation site The C terminal domain of the largest subunit or RNA pol 2 undergoes extensive phosphorylation prior to transcritiption elongation TF2B has a role in converting closed-promoter into an open promoter complex Multiprotein complex invovled in communication between upstream control elements and prorteins at promoter

Compare and contrast Hsp60 and Hsp70 protein chaperones

Most folded proteins are not correct, molten globules are different than native proteins in their intermediates. Most of the time it uses molecular chaperones to assist the process from going from intermediate to the final product without accidents. Hsp70 and Hsp60 are big complexes that bind to hydrophobic patches (center of polypeptide)

Explain the purpose of having alternatve splicing patterns

Multiple protein= one mRNA general= exons have different patterns of splicing to create different proteins Expand protein diversity Yield different proteins Depending on cell type, it will splice and process different= neuron or thyroid

Explain how mutations in the splicing process can lead to disease

Mutations that occur at the splice sites can cause diseases= 15% of genetic diseases is defects in splicing Thalassemias, diseases resulting from defective hemoglobin synthesis

Explain role of mediator.

Plays an important role in communicating messages from gene regulatory proteins to RNAP 2. Links upstream regulatory sequences with RNA polymerase 2 and transcription factors at promoter site. Plays roles in activation (conformational change allowing RNA polymerase 2) and repression

Compare and contrast prok mRNA with euk pre-mRNA and euk mature mRNA

Prokarotic mRNA do not have introns and exons. -Pre-messenger RNA contains exons and introns. -Mature mRNA is generated by spliceosomes after modification through addition of a 5' cap and 3' poly A tail. -mautre mRNA is used in translation

compare and contrast prok and euk RNA polymerases in terms of structure and functions

Prokatyotic cells only have one RNA polymearse, wheras eukaryotic cells have three. -RNA polymerases do not require a primer to initate synthesis. -DNA polymerases uses both strands as a template. - RNA polymerases use only defined stretches of DNA as templates.

Describe the step-by-step process and the roles of all the proteins/ complexes involved in transcription initiation for RNA polymearse 2

Promoters= Tata box and initator, or if you dont have a Tata box you have an initaitor and a downstream promoter element enhancers are upstream -extreme fast -TATA box= between -30 and -100 nucleotides -INR- located btwn basepairs -3 and 5 -if no TATA, DPE, located between 28 and 35 -regulatory elements upstream= CaAT and GC box SUMMARY -Sequential assembly of TBP (TF2B, TF2F, Pol 2, TF2E and TF2H) results in a closed complex -DNA is unwound at he Inr region by helicase activity of TF2H and TF2E creating an open complex -Carboxyl terminal domain of largest Pol2 subunit is phosphorlyated by TF2H -Polymerase escapes promoter and begins transcripion -Elongation is accompanied by release of many transcription factors and is enhanced by elongation factors -Pol 2 released after term order of binding= TF2D/TBP->TF2A->TF2B->TF2F/RNA pol2-> TF2E-> TF2H

Explain how ubiquitin targeting and the proteasome work to degrade proteins

Proteasome pathway has proteins tagged by polyubituqitnation to degrade the proteins- preventing build up of the aggregates -ubiquitin gets covalently linked to its target via isopeptide bond, which marks protein for degradation -Ubiquitin-acivation enzyme adenlyates Ub and transfers to a cytesine residue on E1 -Proteasome recognizes that ubiquitin target

Western blot

Protein expression

ELISA

Protein expression= express gene of interest in model systems= purify proteins and examine them under different conditions. Put protein of interest in vectors to let e coli express protein of interest.

Describe the process and purposes of mRNA degradation and mRNA editing

RNA editing- change the sequence of the mature RNA

Explain why aminoacyl-tRNA synthetases are very specific and have high fidelity

Responsible for attaching the specific amino acids onto the correct tRNA molecule Aminoacyl tRNA synthetases can be divided : Class 1 and 2 aminoacyl bind to different faces of tRNA -Class 1 add to 2'OH, class 2 add amino acid to 3' OH -specific binding site for atp and amino acid -this ensures that the correct amino acid is attached to the trna -specificitiy is attained by various means in different enzymes (proofreading = editing site and an active site, the arm is flexible and it can flip from site to site)

Describe how SRP works

SRP recognizes the signal sequence, and will bind and halt protein synthesis. SRP receptor will deliver the ribosome to the membrane. Then the translocon can go from closed to open, allowing the ribosome to thread through the membrane.

Chromatin structure and modifications can affect transcription

Several factors (chromatin remodeling, histone modifications) Repressed= methylation of histone Heterochromatin is tightly pakced and condensed, mainly due to protein-protein interactions Euchromatin allows transcription to occur because it is packed a lot less densley. Histone can activate transcription by reducing affinity of histones for DNA (methylation of histones), recruiting other componenets of transcriptional machinery and 3) by initating the remodeling of the chromatin structure.

Define ribozyme

Some RNA are catalysts, they self-splice. Self splicing or group 1 introns can excise themselves 3-D structure= integral to function Inactive if denatured Saturable, active site, can be inhibited Nucleophilic attack of sugar OH on phosphate followed by phosphodiester bond hydrolysis

Nuclear hormone receptors regulate transcription

Steroid hormones are regulatory molecules. Estradiol controls sex characteristics, effective because it binds to the estrogen receptor, which is a nuclear hormone receptor. These are acrivated by binding small molecules or ligands. Estrogen receptor binds to the ERE. They have two domains: DNA binding domain and ligand binding domain. Ligand binding causes a strucutral change that enables the receptor to recruit other proteins that regulate transcriptoin (coactivators). The coactivator stimulates transcription. When not bound, they can bind to corepressors and inhibit transcription. These receptors can be tagrets for drugs. Agonists activate, Antagonists inhibit. Cancers are dependent on the estradiol-receptor complex, tamoxifen and raloxifene are receptor antagonists.

Compare and contrast the detailed steps of protein synthesis in proks and euks

Termination= elongation continues until a stop codon appears. Recognized by release factors bound by hydrogen bond. Water attacks the tRNA and polypeptide making an empty tRNA in the P site. Ef-G and Ribosome release factor bind to catalyze the release of the ribosome. Use 4 ATP equivalents for each protein= 2 for activation, 2 for elongation Rapid process due to polysomes MOST steps are identical besides initiation, eukaryotes are more complex -ribosomes are larger in euks because tehy have the 40s and 60s -no formylated methionine, rather requires an initator tRNA -scan along the mrna from the 5' cap until hits the AUG (no shine dalgarno sequence) -eIFs -mRNA is circula because of interactions between the 5' cap and poly A tail

DNA looping

The DNA can bend and loop which brings the binding site and promoter site into close proximity, allowing the thousands of bp away to be there

trans-acting elements (transcription factors) enhancers

These bind to proteins and regulate polymerase activity

Chromatin immunoprecipitation

Transcription factors. ChiP-sequencing analyzes protein interactions with DNA. ChIP-seq is a powerful method for identifying genome-wide DNA binding sites for transcription factors and other protiens. DNA-bound protein is immunoprecipitated using a specific antibody. Gal4 binding sites in yeast= zinc finger interactions= ChiP-seq= find sequences availible for transcription= all transcription factors are bounded to chromatin= only the gal 4 bound to antibody, affinity chromatograhy

DNA footprinting

Transcription factors= Find a DNA binding site on the DNA. When DNA is bound by protein it is protected from chemical cleavage. Isolate a DNA fragment thought to contain a binding site, radiolabel the DNA. Used to find the lac operon = RNA polymerase binding to the lac promoter. 5' end of the nontemplate strand lableed

Describe the function of an activated carrier

Transfer a molecule from one to another

Describe how a helix-turn-helix motif works, and compare it to other, less common DNA-binding motifs found in proks

Two helices combine with a turn in the middle. Functions as dimers, 34 bp apart. Bind in two consecutive major grooves, two major binding sites. First is recognition sequence, then there is a second helix.

Anabolism

Use ATP and reducing power to synthesize large biomolecules- build

Descrbie the processes in the first three stages (activation initiaoin and elongation) in bacterai

activation- tRNA is activated using aminoacyl tRNA synthetases = releases pyrophospahte, driving ligation, then the trna is added to the amino acid Initiation- mRNA aminoacylated tRNA binds to ribosome, (polycistronic, each binding site translated by separte ribosomes- happens 25 nucleotides from 5' end) Ribosome binds to shine dalgarno sequence Initated by a formylated methionine, gtp hydrolysis required) Elongation- Cycles of amino acy binding until a stop codon reached -Ef-TU (elongation factor) bound to aminoacyl tRNA having amino acid, delivered to A site (GTP hydrolysis) in order to finish elongation, must free A site, (charged trna in a site, catalyze peptide with ef-tu)

Describe the structures and functions of tRNA, aminoacyl tRNA synthetases and aminoacyl tRNA molecules

adapter molecules = cca arm and anticodon at bottom aminoacyl trna synthetase= catalyze rxn bacterial RNA polymerases do not cap, only rna polymerase 2 has a cap and it does not make trna

activators

bind enhancer sites which increase transcription= can be thousands of bp away from promoter and still be effective

DNA microarrays

mRNA expression. Allows the determination of the expression pattern of a large number of genes. Allow the determination of the expression pattern of genes simultaneously. Fluorescently labeled cDNA is hybridized to the microarray to reveal expression level of each gene on chip. Use microarrays to detect gene expression changes in cancer cells.

Quantititave PCR

mRNA expression. Detect mRNA before translated or degraded = euks. PCR= double stranded DNA separated through denaturation, cooled to anneal primers, add dNTPs. Repeat multiple times until there are exponential amounts of products. PCR on cDNA allows amplification of coding sequence= isolated and converted into cDNA and that is used as the target for PCR. Determine the quantity of individual transcripts in the cell. Use dye that will bind to double stranded DNA, interculate between strands= more mRNA with a higher number. Measure individual mRNA compared to a housekeeping gene= figure out which is overunderexpressed.

in situ hybridization

mRNA expression= reveals when and where a gene is expressed. In situ hybridization, gene of interest is replaced with a reporter gene. Start with DNA molecules, coding sequence, replaced with reporter gene, make sure you get same expression pattern.

Comapre and contrast prokaryotic and eukaryotic transcription processes for elongation and termination of transcription

mRNA in bacteria is not proccessed -multicellular organisms use differentital gene regulation to generate different cell types -eukaryotes having a nucleus process mature mRNA into the cytoplasm for translation termination= prokaryotic has rho dependent and independent processes eukaryotes= clevage sequence is recognized, poly A addition, endonuclease is part

List how eukaryotic mRNA is processed from pre-mRNA to mature mRNA

pre-mRNA is capped immediately, spliceosome splices out introns, termination and adding a poly A tail -Dozens of proteins coordinate -splicing -adding a cap= as soon as the mRNA comes out, no consenssus sequence= all RNA that comes out of RNA polymerase 2 will have a cap= protects from phosphatases and nucleases -adding a poly A tail -mRNA degradation

Describe how regulatory RNAs are procesed in eukaryotes

pri-micoRNA, cleaved twice ti mature microRNA, boundby argonaute protein 1)Long precursor cleave drosha 2)exported and modified using dicer into dsRNA 3) complement of miRNA removed by helicase 4)partially bound- slow down translation 5) if perfect, mRNA is cleaved totally

Describe how rRNA is processed

rRNA= rlnr is a promoter that lies upstream of initiaton site- upstream promoter (UPE) lies 150 bp upstream -both recruit rna polymearse 1 pre- rRNA -RNA 1 synthesizes large precursor RNA (45S) processed to yield 18S, 28S and 5.8S -prior to clevage, precursor is altered through modification of bases and riboses, catalysed by snoRNPs are made of snoRNA= base pairs with precursor RNA and does the modifications -rRNA is fast and highly processed rna polymearse produces rRNA, folds and processes, make rna from 5-3

Rho-dependent vs rho-independent

rho independent- riboswitch with FMN or ecoli uses stem loop strucutre to halt rho dependent- rho is a helicase that will bind to specific sequences, crawling up the transcript and it will stop the transcription bubble essentailly, the sequence for recognition is within the transcript itself

Give examples of ribozymes

self splicing group 1 introns (26S rRNA precursor), RNase P (cleaves precursors to tRNAs) Hammerhead ribozyme (cleaves RNA of virusoids) spliseosome

List the start and stop codons for translation in bacteria and eukaryotes

start- AUG stop- UGA, UAA, UAG ( you go away, you area way, you are gone)

Describe the roles that rRNA, tRNA, and mRNA play in protein synthesis

tRNA= 3 binding sites (APE)

riboswitches (transcription termination)

the mRNA will sense small molecules in bacteria itself and halt or begin transcription depending on the amount needed - FMN= premature terminator sequence