1/3/17

Opportunities to communicate/meet with me:

1. Office hours (Thursdays, 8:00 – 9:00 am, 1st floor LSB
study area)

2. After class

3. By email

I will set up review sessions prior to the midterms, dates and
times TBA, keep an eye on the Moodle.

See the syllabus posted online for further informa4on with respect to other policies

1

Control coac4vators RNA processing. d)  Viral internal ribosome entry sites (IRESs): how do viruses ensure that their mRNAs are translated at the expense of cellular mRNAs? Vote on Moodle prior to Midterm 2!! 2 . right? Increasingly the answer to this is changing to “No”. modifica4on. RNAi Sec4on 3: Eukaryo4c transcrip4on accessibility. What you will learn about Sec4on 1: Review and Methods Post-transla4onal Splicing. II & III DNA RNA Protein RNA Polymerase Ribosome Transcrip4on Transla4on DNA RNA Protein Transcrip4on Transla4on RNA Transcrip4on Transla4onal decay. b)  Long non-coding RNAs: Most of the genome is “junk” DNA. DNA Factors. ac4vators. Like a transformer. modifica4on alterna4ve Sec4on 4: Transla4on in prokaryotes Sec4on 2: Prokaryo4c transcrip4on and eukaryotes RNA Polymerase splicing Ribosome Pol I. c)  RNA editing: Changing the sequence of a coding RNA after it has been transcribed means changing the identity of the resulting protein without changing the DNA sequence. and these RNAs can have functions. export modifica4on Sec4on 5: Other elements of RNA mediated gene expression Student choice lecture: (Also know as “An amazing lecture of unbelievable awesomeness”) a)  Riboswitches: RNAs that change conformation in response to binding small molecules that are metabolites in the pathways they regulate. 1/3/17 Course Overview What you should already know…. More of the genome is transcribed than we thought. chroma4n edi4ng.

For example: Testing for the understanding of how RNA interference works (#1) might look like this: 3. The course will also cover several levels of gene regulation that have only been discovered more recently. you should: generate the data that provided us with our current understanding of the mechanisms of gene regulation. placed on the mechanisms of transcription and translation and how these differ between prokaryotes and eukaryotes. 1/3/17 Course Goals: 2.1 •  The enzyme direc4ng transcrip4on is called RNA polymerase 3 .1 Storing Informa4on Transcrip4on Producing a protein from DNA •  Transcrip4on follows the same base-pairing informa4on involves both rules as DNA replica4on transcrip4on and transla4on –  Remember U replaces T in RNA –  A codon is the 3 base sequence that determines what amino –  This base-pairing paWern ensures that the RNA acid is used transcript is a faithful copy of the gene –  Template strand is the •  For transcrip4on to occur at a significant rate. complementary DNA strand that is used to generate the mRNA its reac4on is enzyme mediated –  Nontemplate strand is not used in RNA transcrip4on 3. Understand the nature of the experimental methods that were used to This course has two major objectives. Particular focus will be of hypothetical experiments. Understand the mechanisms by which several important cellular factors for these methods and apply your understanding of these to predict outcomes function in the control of gene expression and regulation. You should be able to describe the rationale 1. By the end of the course.

1/3/17 Synthesis of RNA Transcrip4on Phases γ β γ β α γ β α γ β α α Transcrip4on occurs in The α phosphate is three phases: incorporated into the growing chain 1. which lies just upstream of gene ribonucleo4des in the 5 to 3 direc4on •  Polymerase binds 4ghtly to promoter causing •  Movement of the polymerase along the DNA localized separa4on of the two DNA strands template causes the bubble of separated •  Polymerase starts building the RNA chain DNA strands to move also adding ribonucleo4des •  As DNA transcrip4on passes.  Termina4on 3.13 3.14 Ini4a4on Elonga4on •  RNA polymerase recognizes a region. the •  RNA polymerase directs binding of promoter.  Ini4a4on 2. the two DNA •  A]er several ribonucleo4des are joined together the enzyme leaves the promoter and strands reform the double helix elonga4on begins 4 .  Elonga4on 3.

le] to right •  Analogous to the ini4a4ng ac4vity of •  Transla4on occurs 5 to 3 with ribosomes promoters. there are regions at the other end reading the message 5 to 3 of genes that serve to terminate transcrip4on •  Genes are wriWen so that transcrip4on proceeds •  These terminators work with the RNA from le] to right polymerase to loosen the associa4on between RNA product and DNA template •  The gene s promoter area lies just before the start area. 1/3/17 Termina4on Transcrip4on Landmarks •  RNA sequences are wriWen 5 to 3 . local mel4ng and forming the first few –  Number is the sedimenta4on coefficient - a phosphodiester bonds measure of speed with which the par4cles •  During elonga4on. the polymerase and RNA product protein dissociate from the DNA template 5 . the RNA polymerase links sediment through a solu4on spun in an together ribonucleo4des in the 5 to 3 direc4on ultracentrifuge to make the rest of the RNA •  Each ribosomal subunit contains RNA and •  In termina4on. said to be upstream of transcrip4on •  As a result. the RNA dissociates from the RNA polymerase and the DNA and transcrip4on •  Genes are therefore said to lie downstream of stops their promoters Summary Transla4on - Ribosomes •  Transcrip4on takes place in three stages: –  Ini4a4on •  Ribosomes are the protein synthesizing –  Elonga4on machines –  Termina4on –  Ribosome subunits are designated with numbers •  Ini4a4on involves binding RNA polymerase to the such as 50S or 30S promoter.

1/3/17 Transla4on Adapter Molecule Transfer RNA: Adapter Molecule •  Transfer RNA is a small RNA •  Genera4ng protein from ribosomes requires that recognizes both RNA and change from the nucleic acid to amino acid amino acids •  A cloverleaf model is used to •  This change is described as transla4on from the illustrate tRNA func4on nucleic acid base pair language to the amino •  One end (top) binds amino acid language acid with sequence specific to •  Crick proposed that some type of adapter a par4cular amino acid •  BoWom end contains a 3 base molecule was needed to provide the bridge for pair sequence that pairs with transla4on.18 6 . perhaps a small RNA complementary 3-bp 3.17 sequence in mRNA Codons and An4codons Summary •  Enzymes that catalyze aWachment of amino acid to •  Two important sites on tRNAs allow them to tRNA are aminoacyl-tRNA recognize both amino acids and nucleic acids synthetases •  One site binds covalently to an amino acid •  A triplet in mRNA is called a •  The site contains an an4codon the base-pairs codon with a 3-bp codon in mRNA •  The complementary •  The tRNAs are capable of serving the adapter sequence to a codon found role postulated by Crick and are the key to the in a tRNA is an an4codon mechanism of transla4on 3.

ini4a4ng aminoacyl-tRNA binds •  The ini4a4on codon (AUG) interacts with a special to a site on the ribosome. A site –  At start of message AUG is ini4ator •  This process requires: –  In middle of message AUG is regular methionine –  An elonga4on factor. transloca4on. P site aminoacyl-tRNA •  Elonga4on adds amino acids one at a 4me to the –  In eukaryotes this is methionyl-tRNA ini4a4ng amino acid –  In bacteria it is a deriva4ve called N-formylmethionyl-tRNA •  First elonga4on step is binding second aminoacyl- •  Posi4on of the AUG codon: tRNA to another site on the ribosome. UAA. EF-G euk look for 5’ mRna cap - Energy from GTP Termina4on of Transla4on and mRNA A Summary of Transla4on Elonga4on Structure •  Three different codons (UAG.19 ends define an open reading frame (ORF) 7 . func4ons to aWract ribosomes –  Energy from GTP –  Unique to bacteria –  Eukaryotes have special cap on 5 -end of mRNA •  Second elonga4on step. UGA) cause transla4on termina4on •  Proteins called release factors recognize these peptide bond formation stop codons causing –  Transla4on to stop –  Release of the polypep4de chain •  Ini4a4on codon and termina4on codon at the translocate 3. EF-Tu •  Shine-Dalgarno sequence lies just upstream of the AUG. 1/3/17 Transla4on Elonga4on Ini4a4on of Protein Synthesis •  A]er ini4a4on. requires: - An elonga4on factor.

which are made Mature 5 Cap 5 UTR 3 UTR AAA(A)n-OH mRNA from mRNAs) ORF (coding region) (it is from mature mRNA that one makes cDNA) 8 .20 What are features of coding RNAs (mRNAs)? Eukaryotic mRNAs are transcribed by: RNA Polymerase II 4. Structural Rela4onship Between Gene. mRNA and Protein mRNA and Protein Transcrip4on of DNA (top) +1 A trailer sequence is does not begin or end at present at the end of the same places as transla4on mRNA –  Transcrip4on begins at –  It lies between stop codon first G and transcrip4on –  Transla4on begins 9-bp termina4on site downstream –  This mRNA has a 9-bp –  This mRNA has a 3 - leader or 5 -untranslated untranslated region or a region / 5 -UTR 3 -UTR 3. 1/3/17 Structural Rela4onship Between Gene.2 The Polymerase Chain Reac4on They undergo extensive processing before becoming a mature mRNA +1 Pol II DNA •  Polymerase chain reac4on (PCR) can Promoter Transcription Terminator yield a DNA fragment for cloning exon exon exon AAA(A)n-OH Primary •  PCR is: Poly-A tail transcript intron intron –  More recently developed Splicing no A and Ts in this seq –  Very useful for cloning cDNAs AUG STOP (complementary DNAs.

com/watch?v=v4L7rvmBXbY hWp://www.not normal replication –  Next use forward primer to convert ssDNA to 4. Thermus thermophilus. 1/3/17 Amplifying DNA by PCR Thermostable DNA polymerases come from thermophillic bacteria that live at very high temperatures. lives at approximately 72oC. This makes their polymerases very heat-stable and suitable for the high temperature cycling used in PCR. first discovered in a hot spring in PCRs u4lize three cycling temperatures: Yellowstone National Park. typically) e)  Buffer condi4ons suitable for enzyme ac4vity Where do thermostable polymerases come from? Using Reverse Transcriptase (RT-PCR) in How to clone a cDNA from mRNA using reverse transcriptase cDNA Cloning Important! •  To clone a cDNA from just one mRNA whose sequence is known. For example.youtube.com/watch?v=x5yPkxCLads All PCR reac4ons require: a)  DNA template b)  Primers (two primers flanking the sequence to be amplified) c)  Deoxyribonucleo4des (dNTPs) d)  Thermostable polymerase (Taq or Pfu. use type of PCR called How does a cDNA cloned reverse transcriptase PCR (RT-PCR) from mRNAs differ from the genomic DNA copy of •  Difference between PCR and RT-PCR the same gene? –  Start with an mRNA not double-stranded DNA made by RT PCR –  Begin by conver4ng mRNA to DNA .youtube. Denatura4on: melts the template strands (94-98oC) Denatura4on Annealing: primers anneal to the template (52-70oC) Annealing Extension: DNA synthesized by DNA polymerase (68-72oC) Some helpful PCR movies: Extension hWp://www.12 dsDNA –  Now standard PCR con4nues 9 . Wyoming.

probe attaches to new synth Dna. more fluorescence Gel Electrophoresis DNA Gel Electrophoresis •  Gel electrophoresis is used to separate •  Melted agarose is poured into a form equipped with different species of: removable comb –  Nucleic acid •  Comb teeth form slots in –  Protein the solidified agarose •  DNA samples are placed in the slots 5.1 Molecular Separa4ons This fluorescent-tagged oligonucleotide serves as the O]en mixtures of proteins or nucleic acids are reporter probe quencher generated during the course of molecular –  Fluorescent tag at 5 -end biological procedures –  Fluorescence quenching tag at 3 -end –  A protein may need to be purified from a crude cellular extract With PCR rounds the 5 tag is separated from the 3 tag –  A par4cular nucleic acid molecule made in a reac4on needs to be purified Fluorescence increases with incorporation into DNA product The more mRNA is present. The more template there is. Use this as a PCR template in a qPCR in the presence of a reported probe. the more fluorescence is generated .more probes coming off. 5. the more cDNA is made from the mRNA and the more template for PCR there is. more cDna .1 •  An electric current is run through the gel at a neutral neg Dna move towards pH pos pole 10 . 1/3/17 First: convert mRNA to cDNA using reverse transcriptase.

and proteins of various masses electrophoresis = PAGE) can be separated by gel electrophoresis –  Treat proteins to denature subunits with detergent such as SDS •  Most common gel used in nucleic acid •  SDS coats polypep4des with nega4ve electrophoresis is agarose charges so all move to anode •  Masks natural charges of protein •  Polyacrylamide is usually used in protein subunits so all move rela4ve to mass electrophoresis not charge •  SDS-PAGE is used to separate polypep4des –  As with DNA smaller proteins move faster toward the anode according to their masses 5. the posi4ve pole •  Comparison with standards –  Small DNA pieces have liWle fric4onal permits size es4ma4on drag so move rapidly •  Mobility of fragments are –  Large DNAs have more fric4onal drag so ploWed v.4 11 . 1/3/17 DNA Separa4on by Agarose Gel Electrophoresis •  DNA is nega4vely charged due to DNA Size Es4ma4on phosphates in its backbone and moves to anode. which fluoresces under 5. ethidium bromide. RNAs.2 Protein Gel Electrophoresis Summary •  Separa4on of proteins is done using a gel made of polyacrylamide (polyacrylamide gel •  DNAs.2 •  Same principles apply to RNA UV light when it interchelates between the separa4on bases of helical nucleic acids 5. log of molecular their mobility is slower weight (or number of base pairs) –  Result distributes DNA according to size •  Electrophoresis of unknown •  Largest near the top DNA in parallel with standard •  Smallest near the boWom fragments permits size es4ma4on •  DNA is stained with fluorescent dye.

samples of uses a resin to separate solu4on flowing through the column are collected substances by charge •  Samples are tested for the presence of the protein of Resin is placed in a column and interest sample loaded onto the column •  Proteins with highest affinity material. The rate at which for the resin will require proteins pass through the column buffers with higher ionic will depend on their affinity for strength to compete with the 5. exclude larger ones - Most other molecules flow through without binding •  Larger substances travel faster through the column - Last. charge mediated affinity for Chapter 3) the resin 5-46 Affinity Chromatography Gel Filtra4on Chromatography In affinity chromatography.7 c) Glutathione-S-transferase (GST) for glutathione (i. eluted with imidazole) 5.e 5-47 GST-tag). 1/3/17 Ion-Exchange Chromatography Separa4on by Ion-Exchange Chromatography Chromatography originally referred to the paWern seen a]er •  Once the sample is loaded separa4ng colored substances on buffer is passed over the resin paper + sample •  As ionic strength of elu4on Ion-exchange chromatography buffer increases. eluted with soluable glutathione 5-48 12 .6 the resin. the resin contains a substance to which the molecule of interest has a strong and specific affinity •  Protein size is a valuable property that can be used as a basis of physical separa4on The molecule binds to a column resin coupled to the affinity reagent •  Gel filtra4on uses columns filled with porous resins that let in - Molecule of interest is retained smaller substances. the molecule of interest is eluted from the column using a specific solu4on that disrupts the specific binding Common affinity chromatography methods include purifica4on on the basis of the affinity between: a) an4body and an4gen b) His4dine-tag (H6) for nickel ions (can be cloned onto protein of interest to be purified. (Lehninger 2007.