Bio 3C Exam 4

During transcription, which DNA strand is used as a template for RNA synthesis? Understand the difference between the template/antisense strand and the coding/sense strand.

* DNA is the template for RNA synthesis. * Genes may be encoded in either strand. Terminology - Template strand is copied to form mRNA, its sequence is complementary to that of the mRNA. This is also known as the "anti-sense strand". - Coding strand is the other DNA strand, it has the same sequence as the mRNA (with the exception of T's in DNA and U's in mRNA).

DNA is found as a double helix inside the cell, be familiar with the types of interactions that stabilize the helix.

* DNA replication is semiconservativ.e. * Information is in the sequence of bases. * 2 strands held together by hydrogen bonds between 'complimentary bases' that pair according to the 'base-pairing rules'. - A-T & G-C * Base pairing rules allow for the precise transfer of genetic info. We have our parent molecule and then during replication, these strands are copied such that each new daughter cell recieves one parental strand and one new strand (in pink). Macro-Structure of Nucleic Acids * Nucleic acids are a linear polymer of nucleotides. * Nucleotides are covalently bound together by a phosphodiester bond. * Sugar phosphate backbone vs. bases. * Yellow is the nitrogenous base, pink is the sugar, and blue is the phosphate group. This pattern repeats each time. We can look at the pink and blue portion and call it the sugar phosphate backbone, and we can contrast that with the yellow nitrogenous bases. 3 hydrogen bonds hold Guanine and Cytosine together. 2 hydrogen bonds hold Adenine and Thymine together. * 1 negative charge at each phosphodiester bond. * Negative charge repels hydroxyl ions that could hydrolyze the phosphodiester bond. Here, we're looking at the sugar phosphate backbone of RNA and DNA. RNA has that 2' hydroxyl group on the sugar (sugar ->phosphate->sugar->phosphate etc.). DNA has the same sugar->phosphate pattern. But, the 2' C has a hydrogen making a deoxyribose sugar. And, the nitrogenous bases for DNA are either A,T,C,G. RNA nitrogenous bases are: A,U,C,G. DNA has an overall negative charge (remember electrophoresis). 1 strand of DNA * Nucleic acids have a chemical direction/polarity. * 5'-PO4 vs. 3'-OH 5' end is called the 5' end because it terminates with a phosphate group attached to the 5' carbon. In contrast, the 3' end terminates with a hydroxyl group at the 3' carbon. Nucleoside triphosphates (NTPs) are the building blocks of nucleic acids. * dNTPs refer to deoxynucleotide triphosphates (DNA). As nucleic acid strands are being built (during DNA replication and transcription). Nucleoside triphosphates are used during transcription, dNTPs would be used during replication. DNA (or Nucleic Acid) Sequence * ACG (pink) * pApCpG (purple) * pACG (light purple) * Always written 5'->3' There are a variety of ways to show the nucleic acid sequence. * DNA forms a right-handed double helix. * The strands run in opposite directions. * 10 nt per turn. * Each base is separated by 3.4 Ao. (rise) * 34 Ao per turn (pitch). * Diameter of the helix is 20 Ao. * Consistent diameter because a purine always base-pairs with a pyrimidine. We know that two helical DNA strands are coiled around this common axis shown in grey. The strands are parallel, but they're anti-parallel. * Axial view * Double-helix is stabilized by: 1. Hydrophobic effect, driving bases to the interior of the helix. 2. Van der Waals forces between neighboring bases, an attractive force; base stacking. Rotation of about 36 degrees per base and the bases are stacked on eachother. Recall H-bonds hold the purines and pyrimidines together.

Differentiate between a glycosidic bond and a phosphodiester bond.

* Nucleotides are covalently bound together by a phosphodiester bond. * The bond present between sugar and nitrogen base is the glycosidic bond. The bond which joins or connects the phosphate group with the sugar molecule is known as a phosphodiester bond. * The type of bond that holds the phosphate group to the sugar in DNA's backbone is called a phosphodiester bond. Hydrogen bonds connect bases to one another and glycosidic bonds occur between the sugar groups and the base groups.

Be able to identify the major and minor grooves of DNA and why this is important.

B-DNA has major and minor grooves where bases are accessible. Proteins that bind specific DNA sequences interact with these grooves. * Hydrogen bonds can be made with exposed residues of bases. * Major groove is bigger at 12 x 8.5 Ao * Minor groove is 6 x 7.5 Ao (width x depth). The pink-white-purple is the sugar phosphate backbone. The orange and yellow is showing us the nitrogenous bases. This is where potential binding sites exist. The glycosidic bonds of the 2 bases in a bp are not diametrically opposite each other. Each base pair (bp) has a larger side (major groove) and a smaller side (minor groove). These grooves are lined with potential hydrogen-bond donors, shown in blue, and acceptors, shown in red. Major and Minor Grooves DNA is a relatively simple molecule: A,T,C,G. Such a simple molecule can result in such complexity because of the proteins that bind to DNA and control its activity. The major and minor grooves are important protein binding sites.

Why is DNA referred to as antiparallel?

Because the strands are oriented in opposite directions.

What processing is needed to synthesize rRNA in eukaryotes and how is this different from the process in prokaryotes?

Chapter 38: RNA Processing in Eukaryotes * Now we're going to zoom in on the arrow here (circled in pink). Virtually all products of txn are processed in eukaryotes. Production of rRNA in Eukaryotes * Txn by RNA Pol I and processing in nucleolus. * Riboses and bases modified. The mammalian pre-rRNA transcript contains the RNA sequences that are destined to become the 18S, the 5.8S, and the 28S rRNA's, and the yellow will become part of the ribosomal subunits.

What is the importance of the CTD of RNA pol II and how is it modified during the initiation/elongation phases of transcription? What are the functions of the CTD of RNA Pol II?

Eukaryotic RNA Polymerase * Large proteins with different subtypes that have 8-14 subunits. * All are homologous to each other and prokaryotic RNA pol. * RNA pol II (makes the strands that become mRNA) is unique: it's regulated by phosphorylation. - Contains a 220-kd carboxyl-terminal domain (CTD) consensus sequence with multiple repeats of YSPTSPS. - Serine residues "S" phosphorylated (regulation). - Phosphorylated residues bind other factors. * Utilize different promoters to transcribe different RNAs Phosphorylation of the CTD will enhance transcription and recruit other factors required to process the RNA pol. II product. Initiation of Txn in Eukaryotes So, here we've got TFIID which includes the TBP that binds directly to the TATA box. Next, we have TFIIA followed by TFIIB, and then TFIIF, RNA polymerase shown in yellow, TFIIE, and TFIIH will bind the complex, one after the next. TFIIH opens the DNA double helix and phosphorylates the CTD, the carboxy terminal domain. This allows RNA polymerase to leave the promoter and to begin transcription. Phosphorylation of this CTD by TFIIH marks the transition from initiation to elongation.

How are the characteristics of eukaryotic promoters different from prokaryotic promoters?

Promoters: Prokaryotic vs. Eukaryotic Prokaryotic Promoters * The -35 region must be on the coding strand to be effective. * -10 and -35 regions are binding sites for the sigma factor. Eukaryotic Promoters * CAAT and GC boxes on the coding or template strand. * TATA, CAAT, and GC boxes are not bound by RNA polymerase but by other proteins = transcription factors. - Transcription factors help RNA polymerase bind. * Enhancer sequences: 1000s of bps away from the start site of txn (+1).

Understand how trinucleotide repeats may be expanded during replication.

Triplet Repeat Expansion * Can occur during replication if the newly synthesized strand loops out; after replication this strand is longer than the template strand. * Occurs in Huntington Disease. - 6-31 repeats is normal while 36-82+ results in disease (Huntington Disease is an example of this, in a particular section of chromosome 4). Last chapter we learned about the looping out of the lagging strand, which is just a natural process. All of us have sections of DNA with repeats, including these trinucleotide repeats we see here. During replication, a section can loop out and that doesn't have any effect on the reannealing of the remaining sequence. This loop can be small and have no effect, but, as it becomes longer it does have an effect.

Understand what type I and type II topoisomerases do.

Type I topoisomerases relax supercoiled DNA, thermodynamically favorable (no ATP required). Type II topoisomerases (called DNA gyrase in bacteria) use ATP to add negative supercoils, compacting DNA.

What is the composition of RNA polymerase holoenzyme found in E. coli?

α2ββ'σω * Note that there are two copies of the alpha subunit.

What combination of transcription factors makes up the basal transcription complex?

* TFII's along with RNA polymerase II form the Basal Txn Apparatus

What is a polycistronic message?

* mRNA strands derived from operons are called polycistronic mRNA. * Several structural genes are transcribed as a single mRNA molecule = polycistronic transcript or polycistronic mRNA. * Purpose - express operon only when the cells need it. (i.e. low glucose and lactose present / low Trp levels).

How does DNA polymerase ensure that the correct, complementary base is added during synthesis of DNA?

E. coli DNA Pol. I, Klenow fragment * Palm has polymerase activity. * Has a second active site for exonuclease activity to remove incorrect nucleotides. * 3' to 5' exonuclease activity allows for proofreading. * 5' to 3' exonuclease site not shown. This is a closer look at the protein structure Pol. I. It is special because it was the first polymerase we discovered. It also goes by the name "Klenow fragment". It resembles the shape of your right hand. The finger and thumb domains in pink and blue are going to wrap around the DNA, just like your hand would wrap around a baseball bat. * It can go in both directions: 3' -> 5' or 5' -> 3' end. Shape Selectivity by DNA Pol. Increases Fidelity * Binding of dNTP triggers a conformational change. * DNA pol. clamps down on template base and dNTP. * Tight pocket only accommodates complementary bases. * Bond is formed after conformational change occurs. DNA polymerase has such a low error rate. This is possible because: one a dNTP enters into the active site, it triggers a conformational change. Notice the alpha helix really moved. So now, the DNA polymerase will clamp down on the template base (yellow) and the black dNTP, and it forms a pocket. This pocket is really tight and this is where that phosphodiester bond will be created. This tight conformation is only possible when the dNTP corresponds to the watson-crick partner of the template base. So, the template being here in pink, and we'll be adding on this new black dNTP. If we have a pyrimidine and a pyrimidine, we won't get this tight pocket forming. Fidelity of DNA replication * Overall error rate of replication < 10⁻⁸ per base pair. - i.e. less than 1 error for every 100 million nt added. * Incoming bases are most energetically stable following the base pairing rules, or watson-crick pairings, stabilized by hydrogen bonds. Watson-crick pairings help to make sure we have that tight pocket. * Induced fit between enzyme and complementary pairing between the template base and incoming dNTP. * Proofreading by DNA polymerase. * Mismatch repair systems also exist - other enzymes involved. Hydrogen bonds form between the watson-crick pairs: A-T, C-G, that's helping to keep that very stable tight pocket. That conformational change is an induced fit. Even if the base pairing ends up being incorrect, DNA polymerase has proofreading capabilities and mismatch repair system. Proofreading by DNA Polymerases * E. coli DNA Pol. I can remove mismatched bases from 3' end. * Weak H-bonding between mismatched bases, the newly added nt flops around due to Brownian motion. * Polymerase will pause and nt enters exonuclease active site. So we mentioned that tight pocket for DNA polymerase to ensure watson-crick base pairing, if the wrong base pair is added, what happens next? The growing nucleotide chain will occasionally leave the polymerase site, in yellow, and this would especially happen if there's an improper base pairing. That growing chain will migrate into the active site of this exonuclease and here, one or more nucleotides can be exised from the newly synthesized chain. So, this activity is ongiong. Again, it's that weak H-bonding between mismatched bases that lead to that migration into the active site. The polymerase pauses, the nucleotide enters the active site of the exonuclease, it gets cleaved, and then polymerase tries again.

Understand the structure of chromatin in regard to nucleosomes and histones.

Indian muntjac deer has 3 pairs of chromosomes, 1 over 1 billion bp. * Compare the deer's chromosome (red) to a pair of human ones (green) (Basically the deer's were way longer). DNA molecules are linear and they can be very long. TAKE HOME: there's a wide variety in the length of chromosomes. Compaction of DNA in Cells * E. coli has 1 circular chromosome, 4.6 million bp long. - Full extended, its DNA would be 1000x as long as the cell. * Humans (diploid) have 23 pairs of chromosomes for a total of 6 billion bp of DNA. - 3.6 meters long laid end-to-end. * The double-helix itself can be twisted, called supercoiling. * Eukaryotic DNA is bound and organized on proteins called histones - like thread wrapped around a spool. * DNA associated with histones is called chromatin and makes up eukaryotic chromosomes. How can all that DNA fit into a little cell, or for eukaryotes, into an itty bitty nucleus? Supercoiling solves this problem by forming a much more compact structure. Histones assist in the coiling. Electron micrographs of circular, mitochondrial DNA A. Relaxed B. Supercoiled * DNA Duplex in B Form: 260 bp DNA sequence, relaxed 10.4 residues per turn = 25 turns (260/10.4 = 25) * 2 fewer turns and 2 supercoils. This figure describes DNA supercoiling. How can DNA coil so that it fits really tightly inside of a cell? And inside of a nucleus in a eukaryotic cell? We can join the ends to produce a relaxed circle. But, this confirmation still takes up a lot of space. What if we took that linear DNA but unwound it by two turns so that we have 23 turns rather than 25? We can then join the two ends to give us an unwound circle, and that leads us directly to a negative super helix. Summary: the partial unwounding of (D) allows the DNA to take on a superhelical form. We have enzymes - topoisomerases - that facilitate this unwinding and winding to lead to superhelices. This happens in both circular and linear DNA. Negative Supercoiling * Negative supercoiling means it's a right-handed turn. * Most common. * Provides a compact shape, yet it is easy for the 2 strands to be separated. * Circular or linear DNA can be supercoiled. * DNA sequence may be relaxed or supercoiled; these 2 different forms are referred to as topoisomers. * Enzymes that relax or supercoil DNA are called DNA gyrases (in bacteria) or topoisomerases. The same DNA sequence that's taking on different forms such as relaxed versus supercoiled: topoisomers. The enzymes that relax or supercoil the DNA are called topoisomerases, or DNA gyrases in bacteria. Chromatin - Beads on a String (Eukaryotes) DNA in our cells is highly associated with protein. The DNA with protein together is called chromatin. There's the DNA strand, and then it loops around protein complexes called nucleosomes. Nucleosome Core Particle Components * Histone Octomer (made of 8 proteins) = (H3)2(H4)2 tetramer and 2 H2A-H2B dimers. * (B) Histone tails (at Lys and Arg) can be covalently modified to alter chromatin structure. - These tails are really important in controlling the relaxing or supercoiling of DNA. Why do we need to open up DNA? Well, we're opening it up all the time to allow for transcription to take place. But, that has to be shut down then quickly as well. Those histones and their tails are really important for that process. Histone H1 H1: On the outside of the nucleosome core particle. So, if you say core, you're talking about the octomer. If we talk about the nucleosome, that includes H1 and that's here at the entry and exit point of DNA from the nucleosome. In fact, half of the mass from a eukaryotic chromosome is just histones. So, this is just one nucleosome, and it turns out there are higher levels of structure when we talk about chromatin. * Higher-order chromatin structure. * Chromatin itself folds into a helix of stacked layers, 6 nucleosomes per turn. * 1 Ao = 0.1 nm Each of these blue balls is one nucleosome. The chromatin will fold itself into a helix of stacked layers with six nucleosomes per turn. You can see with this higher order structure, you can really compact that DNA down. Eukaryotic DNA - Relaxed to Compact * Metaphase chromosomes are the most condensed and are compacted 10⁴x, as compared to relaxed DNA. We see the double-helix DNA, but it winds around nucleosomes so that we see them as beads on a string, and these nucleosomes form a more compact structure, and we call this structure the 360-Ao fiber. And then, from there, that packs even more to higher order chromatin, and that becomes the chromosome that we can see using a standard microscope. DNA is constantly being opened and closed repeatedly for transcription in little sections. Replication happens once in a cell so that one cell can divide. Transcription, opening and closing that DNA for genes to be transcribed and then translated, that is happening around the clock all the time throughout that cell's lifespan. Cisplatin: Cancer chemotherapeutic * Disruption of the structure of DNA blocks the use of DNA. * Cells can't complete replication or txn. and die. Our understanding of DNA structure has helped us develop drugs to target cancers. Cancer cells grow and divide much faster and so chemotherapies often target DNA and kill those fast growing cells, which is what we want to do when treating a tumor. Cisplatin binds to DNA and disrupts its structure, and then that disruption leads to cell death. The chloride ligands are displaced by purine nitrogen atoms on two adjacent bases, and the cell cannot complete replication or transcription and so it dies: apoptosis. Apoptosis is a good way for a cell to die because if a cell just bursts open, it causes a lot of inflammation and damage to the surrounding tissues, but when the cell begins the process of death through damage of the DNA, that process results in apoptosis which is a very clean way for that cell to die without damaging surrounding tissues.

Understand how the lac operon is controlled by a repressor and CAP proteins.

Lactose is an alternate fuel source, other than glucose * Catabolism of lactose requires 3 enzymes: 1. allow lactose into the cell, permease. 2. cleave disaccharide (lactose), Beta-galactosidase. 3. thiogalactoside transacetylase will detox other molecules transported by the permease. To understand how the operon works: "Why would an E. coli cell use lactose?". Alternate nutrition source. * This reaction is a hydrolysis rxn. Lactose -> galactose and glucose * galactose enters at step 2 of glycolysis after being converted into G-1P and then G-6P. * glucose enters into step 1 glycolysis lac operon expression increases with presence of lactose If beta-galactosidase is grown on a sugar like glucose, one E. coli cell will have a very low amount of B-gal at any given time. Once we add lactose, we see much higher amounts of B-gal being produced. If lactose is removed, then there's no increase in B-gal any longer. If you give E. coli both lactose and glucose, it would rather use all the glucose first before it turns on the lac operon. E. coli have a way to sense the presence of glucose to continue using it if it's available. Regulation of the lac operon * Gene expression is highest when (1) lactose is present and (2) glucose levels are low (txn is controlled). * Uses 2 transcription factors, a repressor and an activator (CAP) to enable cells to sense these 2 conditions. 1. Repressor binds DNA to repress Txn. - When lactose is present, some are converted to allolactose, which binds to the repressor (encoded by gene i), changing its conformation (of repressor protein) so it no longer binds DNA. 2. CAP binds DNA to activate binding of RNA pol. (increase Txn). - When glucose levels are low, cAMP levels rise. cAMP binds CAP allowing it to bind near the promoter. Repression of the lac Operon We're looking at the operon in the absence of lactose. Gene i encodes imRNA and that has the message for the repressor proein shown in purple. The repressor protein binds directly to the operator. In doing so, it blocks RNA polymerase from moving to the right and transcribing z, y, and a. Induction of the lac Operon In the presence of lactose, some of the lactose is converted into 1,6-allolactose. Allolactose is shown in pink and it binds directly to the repressor protein. In doing so, it changes its shape so that it cannot bind to the operator. With nothing blocking RNA polymerase here at the operator, RNA polymerase can move to the right and transcribe z, y, and a. Recall we get this polycystronic strand of mRNA and that is then translated into 3 distinct proteins. That's Beta-gal, permease, and transacetylase. If I need allolactose to permit the production of B-gal, hwo did we get allolactose in the first place? Inducers Bind The Repressor to Induce Txn Allolactose produced by a side reaction of B-galactosidase (which cleaves lactose into glucose and galactose). * Comparing lactose with allolactose. * Lactose has an alpha-1,4 glycosidic linkage. * Allolactose has an alpha-1,6 glycosidic linkage. Lactose converted to Allolactose * Allolactose produced by a few molecules of Beta-galactosidase that are present before induction of lac operon. It's important to know that proteins don't bind to DNA with superglue. Instead, there will be moments when the lac operon is transcribed, even in the absence of lactose and allolactose. Allolactose is produced by a few molecules of Beta-gal that are present even before the induction of the lac operon. Activation of lac operon * CAP is the activator protein: Catabolite Activator Protein * CAP only binds the DNA when cAMP levels are high. * cAMP + CAP binds DNA. * CAP recruits RNA Pol to the promoter to increase Txn rates. * When glucose is increasing cAMP is decreasing and thus glucose inhibits the lac operon = catabolite repression. CAP has a binding site for cAMP. CAP proteins bind at about -61 from the transcription start site. CAP is needed to help RNA Pol bind to the promoter. CAP will only bind at -61 if it's bound to the cAMP. cAMP is only produced when glucose levels are low. If glucose levels are high, then there won't be enough cAMP available to bind to CAP to allow it to bind here to the DNA at -61. If glucose is available, why bother transcribing z, y, and a? Control by CRP (=CAP) A. cAMP binds to CAP, which is otherwise inactive. In doing so, it makes CAP active. It then binds here at the promoter at about -61, relative to the txn start site. CAP then facilitates RNA Pol binding to the promoter, and moving to the right to transcribe z, y, and a. B. cAMP levels will be low if glucose is present. So, cAMP won't be avilable to activate CAP. Without CAP, RNA Pol. is less likely to bind and we won't have transcription. The repressor protein won't bind to the operator because it's bound to allolactose. Nonetheless, RNA pol can't transcribe because it doesn't have the help of CAP. Lac. operon: the lactose operon contains three genes. Transcription is controlled by regulatory proteins that bind to an activator-binding site and an operator. Glucose present, no lactose: Transcription not activated AND blcoked. * Low cAMP; CAP cannot bind. In addition, repressor is bound to operator, blocking polymerase. Glucose present, lactose present: Transcription not activated. * Low cAMP; CAP cannot bind. At the same time, inducer (allolactose) prevents repressor from binding to the operator. No glucose, no lactose: Transcription activated but blocked. * High cAMP; CAP/cAMP complex binds to activator-binding site. Repressor is bound to operator, however, blocking polymerase. No glucose, lactose present: Transcription activated. * High cAMP; CAP/cAMP complex binds to activator-binding site. In addition, inducer (allolactose) prevents repressor from binding to the operator.

What is an operon? Is it found in prokaryotes/eukaryotes/both?

Regulation of Gene Expression in Prokaryotes: lac Operon Our very first peak into how gene expression is regulated ocurred in E. coli and specifically, with this set of genes that we now call the lac operon. So, before we discovered the lac operon we didn't know how a cell could control whether or not a gene would be transcribed. So, understanding the lac operon is fundamental. Why Regulate Gene Expression? * Energetics - protein synthesis is energetically expensive . Why should I make a protein if I don't need it? * Environmental needs - organism needs to adapt to environmental or physiological conditions. * Developmental needs - multicellular organisms follow developmental plans. - Ex. insects: egg -> larvae -> pupae -> adult Still all the same animal and all the same DNA but wow, these look so different. This is possible because of gene expression. * Different cell types in different tissues have different functions and properties (with the same genome). Possible Levels for Regulation of Gene Expression * Transcription * mRNA processing * Translation * mRNA degradation * Protein degradation Just like a factory that produces a product, you don't want to get to the very last stage and then shut down production. It's much more efficient to control that very first step of product production. Cells have a variety of ways to control transcription in the first place. Transcription Factors = DNA-Binding Proteins * Most common structural motif, a helix-turn-helix protein: fits perfectly in DNA double helix. * Proteins bind DNA as dimers. * Each monomer of the protein inserts into the major groove of the DNA. * 1st alpha helix interacts with the DNA backbone (phosphates). * AA side chains of the 2nd alpha helix make specific interactions with bases, via the major groove. We already know about RNA polymerase binding and the importance of the sigma factor in recognizing the promoter, but RNA polymerase often cannot just bind on its own. It needs the assistance of other proteins. The proteins that also bind DNA and assist in controlling the activity of RNA polymerase are called transcription factors. Gene Organization Unique to Prokaryotes * Operons - First proposed by Francois Jacob and Jacques Monod. - Multiple genes needed for 1 activity are found together in the genome and are regulated together. - Expressed as a polycistronic mRNA (1 mRNA translated -> multiple proteins). * Genetic elements of an operon" 1. Regulator gene, encodes a repressor protein that will bind directly to the operon. 2. Regulatory sequence = operator site. 3. Several structural genes. Operons are not found in eukaryotes. mRNA strands derived from operons are called polycistronic mRNA. Operons Several structural genes are transcribed as a single mRNA molecule = polycistronic transcript or polycistronic mRNA. * Purpose - express operon only when the cells need it. (i.e. low glucose and lactose present / low Trp levels). * Ex. lac operon - Encodes 3 genes needed for the catabolism of lactose. Ex. trp operon - Encodes 5 genes needed for the synthesis of tryptophan. An Operons * p = promoter, where RNA polymerase binds. * i = encodes repressor * o = operator, where repressor binds. We're looking at DNA here.

Where does replication begin in E. coli and how is this similar and different from the initiation of replication in eukaryotes?

Replication Always Begins at the Ori * Origin of replication in bacteria = oriC locus. * DNA begins process to unwind the DNA helix. * 5 repeats for DnaA to bind, DnaA then recruits: - DnaB (helicase), SSB (single-stranded binding proteins). - Makes up the prepriming complex. * Here the strands of DNA are separated and made available for enzymes to copy the DNA The oriC locus has five copies of a particular sequence (yellow). These are binding sites for a recognition protein called DnaA. In green, we see tandem array of 13 base pairs. We call it a 13-mer sequence, and thhese are rich in A's and T's together. So, this is a little bit more unstable than a GC rich region would be. It's easier to open. Once those DnaA molecules bind, the replication complex will be built. DnaA recruits helicase, while also goes by the name DnaB. It also recruits SSBs, the single stranded binding proteins. DnaB will unwind the duplex, SSBs will bind to single-stranded regions, and will prevent reforming of the double-helix. * Initiation of replication in E. coli. * Begins at oriC, one actual site on the chromosome. Replication in Eukaryotes * DNA synthesis occurs during S phase of the cell cycle, followed by mitosis. * Multiple origins of replication exist, each is the site of a replicon. * Formation of DNA synthesis initiation complexes controlled by licensing factors (like prepriming complex). * License expires after 1 use so each replicon is replicated only 1 time. S phase: where DNA is replicated through mechanisms similar to those just discussed. Replication is not an ongoing process in cells, it happens once so that the cell can divide. Permission to divide is controlled by many proteins. Gap period: many proteins control whether the cell has permission to divide or not. When cells no longer wait for permission and just begin dividing out of control, that's now cancer. Replication of Linear Chromosomes - What happens at the ends? Eukaryotes have linear chromosomes rather than circular chromosomes like prokaryotes. This matters because: * Starting here (purple) we see our two replic. forks, and this would happen in both circular and linear DNA. Then here (grey purple) we have our template strand, our newly synthesized lagging strand, the fragments have been ligated together already by DNA pol. III. By DNA ligase the primers are degraded, the gaps are filled in no problem because we always have a 3' end to add onto. At the terminal gap, there's no 3' hydroxyl group, so there is nothing that could be added here to replace the ribonucleotides that were here. So, this is actually an overhang. We call it a 3' overhang, and these are formally known as telomeres. Here (purple) we have our lagging strand with those primers in orange that have to be removed and replaced. This works, but at the end when we remove the RNA primer, we end up with the 5' end, and there is no 3' hydroxyl to add onto. So, this 3' overhang is going to be degraded, and we end up with a shortened template. This is going to keep shortening, which is a problem.

Understand how replication on the leading strand is coordinated with replication on the lagging strand (i.e. the trombone model).

Synthesis on Each Strand is Coordinated. DNA Polymerase III Holoenzyme Consists of two copies of the polymerase core enzyme. These are linked by a central structure that includes something called the clamp loader, which holds the DNA polymerase holoenzyme together by binding directly to the helicase and to polymerase subunits, thereby making synthesis of the leading and lagging strands simultaneous. The sliding clamp is loaded onto DNA by the clamp loader, and that's an ATP-dependent mechanism. Trombone Model explains coordination of synthesis on leading and lagging strands So again we have that clamp loader, and the DNA will loop out, that looping out mechanism allows DNA polymerase to stay here at the fork and still produce those okazaki fragments from the 5' to the 3' direction. That looping out and then it's going to come back closer, we call it the "Trombone slide". Here's (bright green) an okazaki fragment that's already been prouced. Here (bright blue), the primer has been laid down and now pol. III will grow in the 5' -> 3' direction adding on to the 3' end. The template of the lagging strand is called the lagging strand template that will go until it reaches the okazaki fragment ahead of it.

What processing is needed to synthesize functional tRNA in eukaryotes?

tRNA's transcribed by RNA Pol III and modified From the early transcript, the 14 nt intron shown in yellow is removed. This UU at the 3' end is removed, the leader sequence is also removed. Then, bases and riboses are modified, and we end up with the mature tRNA. The CCA (red) is the AA attachment site. At the bottom in red is the anticodon. That will be different for different tRNA's.

Differentiate between snRNA and SNRP.

* snRNP's: strands of RNA combined with proteins, "small nuclear ribonucleoprotein particles". They're the mutli-colored balls. * snRNA's in spiceosomes catalyze the splicing of mRNA precursors. Small nuclear RNAs (snRNAs) are critical components of the spliceosome that catalyze the splicing of pre-mRNA. snRNAs are each complexed with many proteins to form RNA-protein complexes, termed as small nuclear ribonucleoproteins (snRNPs), in the cell nucleus

Understand how the Ames test allows for identification of potential mutagens.

Accumulation of Mutations Can Cause Cancer * Many cancers are caused by defective repair mechanisms. * Cancer involves uncontrolled growth of cells, due to changes in genes related to division and the cell cycle, also genes involved in DNA-repair. - Tumor suppressor genes include genes encoding DNA repair enzymes. If DNA isn't being repaired, that leads to cancer. * Many cancer cells have inactivated DNA repair systems. This allows more mutations to accumulate so the cancer can grow and spread. * These cancer cells can be targeted by drugs that damage DNA. Ex. cisplatin and cyclophosphamide. When we know that DNA repair mechanisms are inactivated, we can use specific drugs. * Cisplatin causes disruption of the structure of DNA and blocks the use of DNA. * Results in cell death if not repaired! Remember, we want a cancer cell to die and to not keep dividing. Ames Test * Identifies potential carcinogens by their ability to induce mutations in bacteria. * Quick, cheap test utilizing Salmonella His- bacteria. - His- indicates this strain cannot make any histidine, due to a mutation in 1 gene for synthesis of His; it can only grow if His is added to the media. 1. Mix a chemical with rat liver extract and expose bacteria. - Enzymes in liver extract mimic metabolism of chemical in the body. 2. Test for the ability of bacteria to grow on media without His. - Mutations may occur that reverse the mutation in histidine synthesis. 3. Lots of growth indicates the chemical is a mutagen. We know that many cancers are caused by exposure to chemicals. In humans, it can be really hard to pinpoint the source. We can look to see if bacterial DNA is mutated and if bacterial DNA is mutated by a potential carcinogen, then we'll know that it's likely dangerous for us. Ames Test for Chemical Carcinogens We start with a test tube that contains salmonella and not just any salmonella bacteria, but a strain that cannot make its own histidine. Since it can't make its own histidine, we have to add histidine to the broth. We also add the suspected mutagen to the test tube (shown in purple), and then in order to mimick the mammalian metabolism,we include some liver enzymes because remember our liver breaks down many toxins. So we put all of this into a test tube and then grow the salmonella on media. Importantly, its going to be media that has no histidine in it. So, remember the salmonella should not be able to grow. If they grow, we call those colonies revertent. They reverted back to being able to make histidine. That's a big deal. Their DNA was mutated to the point where they could make histidine again. Seeing many colonies on a plate tells you that this mutagen is likely carcinogenic. Remember carcinogens are mutagens that lead to cancer. So certainly if you're mutating DNA, that can very likely lead to cancer. So of course bacteria don't get cancer. But, this is just a model organism to help us understand the safety of particular chemicals, and their ability to cause cancer to us. This would be the control tube, again salmonella with rat liver extract, but then without the mutagen. So, when you plate those cells onto a plate without histidine, there shouldn't be any growth. Why do we see two colonies? Well, bacterial DNA mutates, and we have what are called spontaneous revertants. So, there will be some growth. Bacteria divides so quickly and mutations do arise. So, in these two colonies, yes they were able to make histidine again. But importantly, that's contrasting with the plate prior where we saw many more colonies. Ames Test There are actual petri dishes using a suspected mutagen. Notice all of these revertant bacteria growing around the disk (B). All of these salmonella colonies reverted back to being able to make histidines. How did they change their DNA? Well that was from the mutagen.

DNA can be damaged in many ways. Know what happens as a result of: oxidation, deamination, alkylation, and UV light.

Chapter 35 - DNA Repair and Recombination From UV light, thymine dimers can form. Sometimes they're repaired, but sometimes they're not. Other sorts of DNA damage can happen spontaneously. A wrong base can be incorporated, chemicals can modify bases, there can be chemical crosslinks such as the thymine dimers, you can also have breaks in the phosphodiester backbone. The result of DNA damage would be cell death or cell transformation. So, the cell is going to behave in ways it didn't act before. Oxidation of Bases Leads to Wrong Pairings * OH- radicals can oxidize bases. * A G-C base pair becomes a 8-Ox-A base pair, and after replication is a T-A base pair. - Can result in a point mutation where one base is changed. Will lead to the wrong pairings. Initially, it's the correct base pair, but if one of the bases becomes oxidized, it will base pair with the incorrect nt. Deamination of Bases also Alters Base Pairing * A-T bp becomes a hypoxanthine-C bp. * After replication it is a G-C bp. Hypoxanthine bp with cytosine. A-T bp becomes a G-C bp after replication. Alkylation * Hydrocarbons can be added to bases, also altering base-pairing properties. * Ex. Mutagen is aflatoxin (made by mold growing on grain or peanuts), it becomes mutagenic after metabolism in the liver. * Can react with guanine. * G-C base pair can become a T-A base pair. - The modified G pairs now with A. Upon metabolism in the liver, aflatoxin epoxide will react with DNA. This aflatoxin epoxide is highly reactive and will alkylate guanine, changing it from a G-C bp to a T-A bp because that modified guanine will pair with adenine, of course G should've paired with C. UV Light (non-ionizing radiation) Another way to damage DNA. Two adjacent thymines can be crosslinked, that's the green lines here. They're interacting in a way that they didn't previously. We call these crosslinks. They can also occur between cytosines as well. These covalently linked thymines will not fit into the DNA double helix properly. So, gene expression will be locked unless this can be removed. * X-rays, which are ionizing can also damage DNA. In the case of x-rays, they induce single and double-stranded breaks in DNA.

Why is DNA replication referred to as semi-conservative?

DNA replication is a semi-conservative process, because when a new double-stranded DNA molecule is formed: One strand will be from the original template molecule. One strand will be newly synthesised. DNA replication is called semiconservative because an existing DNA strand is used to create a new strand. DNA is a double stranded molecule.

Differentiate between mismatch repair, base excision repair, and nucleotide excision repair.

Mismatch Repair * Detects and repairs mismatches that escape proofreading by DNA Pol III. * Several proteins needed: 1. Detect mismatch - MutS Then MutL is recruited. 2. Recruit an endonuclease - MutL MutH is the endonuclease. 3. Endonuclease cleaves DNA near mismatch - MutH. Endo means "within DNA strand". 4. Exonuclease I removes part of 1 DNA strand with mismatch. Exonuclease can work from the ends of DNA. * Methylation distinguishes 2 DNA strands. DNA polymerase III fills in gap. Overall theme: first find the base that's incorrect. Second remove that base. Third fill in the gap. T was the incorrect base and it was replaced with C, the correct base, The repair mechanism knew T was wrong and not G because of methylation. DNA is methylated, but it's not methylated immediately after replication. Instead, newly synthesized strands are not yet methylated. This is how the repair systems know which base was already there. G would've been methylated and the T is not yet methylated on the new strand, and therefore, it's the newer one, and it's the wrong one. DNA is methylated on cytosines, and it's an amazing method of control for DNA activity. Mismatch Repair Summary 1. MutS-detects mismatch. 2. MutL-recruits an endonuclease termed MutH. * "endo" means the endonuclease cleaves phosphodiester bonds in the middle. 3. MuH-cleaves DNA near the mismatch. 4. Exonuclease 1 removes incorrect base and surrounding segment. * "exo" means exonuclease cleaves from the ends of the polynucleotide chain. 5. Polymerase fills in the gap. Recognize - Remove - Repair Base Excision Repair * Removes modified bases, specifically these: - 8-oxyguanine or 3-methyladenine. 1. In E. coli uses enzyme AlkA, a glycosylase that cleaves the modified base from the deoxyribose sugar. This: 2. Creates an AP-site (missing 1 base but the backbone is intact). 3. AP endonuclease cleaves the backbone at this site, on 1 side. 4. Deoxyribose phosphodiesterase cleaves the other side of residual base to remove it. * AP = - A pyrinic: A/G - A pyrimidinic: C/T * DNA polymerase inserts an undamaged nucleotide. * DNA ligase seals the phosphodiester bond. * In eukaryotes cytosines are methylated (adenines methylated in bacteria). * 5-methylcytosine can spontaneously deaminate -> T. * T-G base pair mismatch is recognized that the "T" is wrong by base-excision repair proteins (DNA glycoslyase). If a T-G base pair is recognized, the repair mechanisms know that the T must be the wrong one. The base excision repair proteins that we just learned about will be the enzymes that remove that mismatch. In fact, T-G pairs are so common that T's are just automatically removed by DNA glycosylase. Nucleotide Excision Repair * Back-up system for base-excision repair. * Recognizes damaged bases that are not paired with the 'right' base - distortion in double helix recognized. - Turns out that not all damaged bases are recognized by DNA glycosylase. * Gap filled by DNA pol I and sealed by DNA ligase. Let's say we have a pyrimidine dimer that's distorted, and that distortion is recognized. There's an enzyme called uvrABC exinuclease, exi just means "cut out". So, the dimer and surrounding segments are cut out and then polymerase I will fill in the gap that's shown in pink. And then a phosphodiester bridge created by DNA ligase.

In general, is RNA modified in prokaryotes? Are there any examples of modification of RNA in prokaryotes?

Rare Example of RNA Modification in E. coli * rRNA and tRNA excised ("cut out") from a single mRNA and further modified before they become functional (add nt, modify bases and/or riboses). * mRNA is not modified post-transcription in prokaryotes. Recall we said mRNA is produced directly from the DNA template in prokaryotes. That's true. We would say mRNA is not modified after transcription in prokaryotes. This primary transcript (blue) will be cleaved to give us the 16S strand of rRNA, and the 23S strand and 5S strand of rRNA, and then in pink we see the tRNA that will be cut. Modifications to Bases in tRNA after Txn We see modified adenylates, or uridylate can be modified and methylated so it becomes ribothymidylate or pseudouridylate. CCA is added to the 3' end of tRNA. That CCA sequence will be the attachment site for an amino acid to that tRNA molecule. * Adenylate ---methylated---> 6-dimethyladenylate

Understand the two different mechanisms for termination of transcription.

Termination Signals 1. RNA Hairpin - "Protein-Independent Termination" * RNA hairpin relies on the formation of protein-independent termination. 2. Rho Protein - "Protein-Dependent Termination" * Rho protein relies on the formation of protein-dependent termination. * Both require signals in newly synthesized RNA (rather than DNA template). The termination signal is in the newly synthesized RNA. It's not in the DNA template itself. 1. Self-Termination * mRNA forms a stem loop structure at palindromic repeat. * mRNA dissociates from DNA in Txn bubble. * Txn bubble closes, ending Txn. Hairpin: the end of the mRNA strand forms this stem-loop structure. This is actually a palindromic repeat. This hairpin is stable and is followed by a sequence of four or more uracil residues. RNA polymerase will pause immediately after it synthesizes this section of the RNA trnascript, and this stretch of uracil residues is important because the RNA/DNA hybrid in this section is all A with U. Recall that A's and U's are held together by only two H-bonds. So these are weaker relative to C's and G's. So, the pause plus this weak association results in the dissociation of the DNA template from RNA polymerase. Then, the txn bubble closes and txn ends. 2. Protein Dependent (Rho, ρ) Termination Second method of termination of transcription. A protein called Rho (ρ), in this case, a helicase, moves up the nascent RNA strand and it pulls it away from RNA polymerase and the DNA template. So again, with these two methods, either protein dependent like we see here, or protein independent (hair pin), the termination signal itself lies in the newly synthesized RNA rather than in the DNA template.

Be familiar with the dimensions of B-form DNA and how it is different from A & Z form DNA.

The Watson and Crick model of DNA shows the form of B-DNA. * A-DNA - Also a right-handed helix, with two antiparallel strands. - Dehydrated, wider and shorter, - Seen in dsRNA, and RNA-DNA hybrids. * Z-DNA - Left-handed helix, flexible. - Z-DNA binding proteins obs. DNA double helices can take on different forms. * B-DNA: most prevalent in physiological conditions. * Z-DNA: left-handed, and the phosphoryl groups in the backbone are zigzagged. Also seen in physiological conditions. * DNA is flexible and dynamic.

Be familiar with the 3 major types of processing that eukaryotic mRNA undergoes (5'-cap, 3'-poly A tail, and splicing). Where in the cell do these modifications occur?

The product of txn is called the primary transcript or the primary RNA transcript. It contains introns and exons. The introns have to be removed, and the exons are spliced together. At the 5' end we have CAP and at the 3' end we have multiple adenylate residues, poly-A tail. We also have, in orange and yellow, untranslated regions, regions that won't be directly used in the production of a protein by the ribosome. 5' Cap added to 5' end of all mRNA's in Eukaryotes * 7-methylguanosine residue joined to transcript's initial 5' nucleotide via 5'-5' triphosphate bridge (added when transcript is about 25 nt long) = cap 0. * May also be methylated at first nucleotide (= cap 1) or second nt (= cap 2). * Function: - capped mRNAs resistant to nucleases. - enhances mRNA translation. CAP is added when transcript is about 25 nt in length. So, shortly after initiation of transcription. This is a 3-step process, so first a phosphoryl group is removed by an RNA triphosphatase. Then, the diphosphate in black at the 5' end of the RNA will attack the alpha-phosphorus atom of a molecule of GTP, and we get a triphosphate linkage. A 5' to 5' triphosphate linkage is catalyzed by guanotransferase. Then, the nitrogen 7 atom of that terminal guanine shown in pink is methylated. The pink here is called cap 0. These next two riboses in black can also be methylated. We'll call the one closest to pink "cap 1" and the next "cap 2". Again, the point of these CAPs is to contribute to the stability of the mRNA, it just is protecting the mRNA from degradation by nucleases and phosphatases, and it enhances mRNA translation. 3' PolyA Tail added to 3' End of mRNA in Euk. At the 3' end, many adenylate residues are added. Here, we have the 5' CAP on our nascent RNA strand. Nascent means it's being newly formed. Then, a specific endonuclease will cleave here at the AAUAAA cleavage signal. Then, a poly A polymerase adds these blue adenylate residues to form the poly A tail. There are usually about 250 of these residues at the 3' end. The donor of those adenylates is ATP. Although we don't know the exact function of that poly A tail, we know that without it, translation efficiency decreases markedly. Overview of Post-Transcriptional Processing of mRNA in Eukaryotes Transcription is followed by a 5' CAP, a poly(A) tail, that primary transcript is then spliced to give us Beta-globin mRNA. That contains exons and the 5' CAP and the 3' poly(A) tail. Eukaryotic Genes Contain Introns and Exons * Bacterial genes are continuous (no introns). * Philip Sharp and Richard Roberts discovered that eukaryotic genes are discontinuous in 1977. - Viewed hybridized mRNA + DNA with electron microscopy. We saw mRNA and DNA didn't align. They observed two annealed sections of DNA with mRNA in a duplex, but there was a loop of unannealed DNA between them, and that was first hint that DNA and mRNA don't have the same length. There wasn't a 1:1 matchup. * Exons - coding regions of mRNA (expressed sequences). * Introns - noncoding regions, spliced out before translation (intervening sequences). The sequences at the ends of introns will specify splice cites in mRNA precursors. They usually begin with AGU and end with AG. The AG is preceeded by a pyrimidine-rich tract, which is considered a consensus sequence. Splicing removes the introns, connects the exons. - Usually begin with GU and end with an AG preceeded by a pyrimidine-rich tract (=consensus sequence for splicing) * Splicing removes introns and connects exons in mRNA. Splicing - why have Introns and Exons? * Many exons encode discrete structural/functional units of proteins (protein domains). * New proteins may come about by rearrangement of exons (exon shuffling). - Means of generating novel genes. * Has the potential to generate a series of related proteins by splicing the primary transcript in different ways (alternative splicing). - Ex. Antibodies attached to the plasma membrane of a B cell versus soluble antibodies that are secreted by the B cell. You have Abs that are secreted into your blood, and you have Abs that sit on the surface of a B cell. Even though one B cell will have Abs with the same variable region, there has to be differences in that heavy chain there at the constant region in order to facilitate attachment of that Ab into the plasma membrane. We've also discovered that introns regulate txn. Often introns contain cis-acting elements like enhancers and sequences we call silencers, they silence txn. So, even though the introns are ultimately removed, they certainly play an important role in our genomes. Directions in DNA Sequence for Splicing 1. 5' splice site (GU) 2. Branch site (A) 3. 3' splice site (AG) Intron is flanked by a 5' splice site with the upstream exon ending in AG and a 3' splice site with the downstream exon beginning with G. In the middle, we see a branch site, "py" just means pyrimidine. We'll actually call that the polypyrimidine tract. So, it contains U's and C's. Transcription and pre-mRNA processing are coupled * CTD (YSPTSPS) of RNA pol II is phosphorylated on serines. * Controlled by kinases and phosphatases. * CTD recruits: * Capping enzymes to methylated 5' guanine during transcription. * Components of splicing machinery. * Endonuclease to cleave transcript at poly(A) addition site. We can see here the txn factor TFIIH, the one that phosphorylates the CTD of RNA pol II. Once that CTD is phosphorylated it signals to the polymerase that its now in the elongation phase. Once that CTD is phosphorylated, it will bind to factors that are required for: capping, splicing, making that poly(A) tail, and those proteins are brought in close proximity to that nascent pre-mRNA. That's the dark green you see here. What we notice here is that it's not just the random bumping in of capping enzymes, and the spliceosome it's not just brownian motion, but really there's a mechanism to bring those important proteins in close proximity with that nascent RNA strand.

Understand what is meant by the term "DNA backbone"?

The sugar-phosphate backbone. Phosphodiester bonds hold the sugars and phosphates together. Macro-Structure of Nucleic Acids Sugar phosphate backbone vs. bases * Yellow is the nitrogenous base, pink is the sugar, and blue is the phosphate group. This pattern repeats each time. We can look at the pink and blue portion and call it the sugar phosphate backbone, and we can contrast that with the yellow nitrogenous bases.

For initiation of transcription in E. coli the σ factor is important. Understand its contribution to transcription and where it binds on the DNA.

Transcription * DNA is transcribed to make mRNA. * Cell components needed for transcription: - Template DNA - RNA polymerase - RNA nucleotides (A,U,C,G) * Types of RNA: mRNA (messenger RNA), tRNA (transfer RNA), rRNA (ribosomal RNA). Transcription (Txn) * 3 major stages 1. Initiation - Promoter sequence (in DNA) marks the beginning of a gene. 2. Elongation - RNA is synthesized by 1 enzyme: RNA polymerase. 3. Termination - Termination sequence (in DNA) marks the end of a gene. Initiation begins at the promoter. Elongation is the process of RNA polymerase adding new complementary nucleotides to the DNA template. Termination occurs at the end of the gene. The Process of Transcription 1. RNA polymerase binds to the promoter, and DNA unwinds at the beginning of a gene. 2. RNA is synthesized by complementary base pairing of free nucleotides with the nucleotide bases on the template strand of DNA. 3. The site of synthesis moves along DNA; DNA that has been transcribed rewinds. 4. Transcription reaches the terminator. 5. RNA and RNA polymerase are released and the DNA helix re-forms. The promoter is upstream of the gene start site. Transcription begins at the start site and RNA polymerase, shown with the "blue bubble" is just adding complementary nucleotides one at a time to the DNA template.That continues until you get to the end of the gene. At that point, RNA polymerase dissociates from the DNA template and the mRNA strand is translated. Here, we're looking at five sequences from different bacterial promoters. What you're taking note of is the sequence in pink. We call that a consensus sequence. That means, on average, we usually see TATAAT in most prokaryotic promoters. Notice, this is here at -10 because the gene begins at one, at the +1 site. In blue is the start of the gene (+1) * Promoter consensus sequence = -10 consensus sequence. Consensus Sequences in the Promoter * E. coli * consensus means average. * -10 Pribnow box (same as -10 consensus sequence) & -35 consensus sequence (bound by σ factor in RNA polymerase). * Strength of promoter helps regulate Txn frequency. * Strong promoters Txn every 2 seconds; weak promoters every 10 min. Genes that have promoters that are very similar or are identical to these consensus sequences are transcribed very frequently. Whereas genes that have sequences that are very different from these sequences are transcribed at a slower rate. The strongest promoters have sequences that correspond most closely to these consensus sequences. Weak promoters have substitution. We won't need to memorize the sequences in pink, but we do need to know they're called the -35 and -10. We should be able to differentiate the -35 and -10 from promoter elements that we see in eukaryotic genes. RNA Polymerase Finds and Binds Promoter via σ Factor RNA polymerase protein, the enzyme. Orange is the sigma subunit, it looks like there are two copies, but there's only one. It recognizes the -10 and -35 element shown in yellow. The sigma subunit is helping RNA polymerase locate the start site of the gene. But, it also decreases the affinity of RNA polymerase for the DNA. This makes sense because we want RNA polymerase to find the start of the gene. But, we want it to also be able to slide along the DNA in order to begin transcription. Initiation of Txn in E. coli We're noticing the multiple subunits of RNA polymerase, (two copies of alpha). The sigma subunit is separate until it combines with the core enzyme at the promoter. Again, its function is to decrease affinity for DNA, so that allows RNA polymerase to slide along the template strand in this figure that's shown in pink. Also, the sigma subunit is there to help recognize the -10 and -35 promoter sequences. Notice that after initiation, we have a small amount of RNA product, and then the sigma subunit dissociates, we say it's recycled, and can be used again by another core enzyme. This open section of DNA goes by the transcription bubble.

How is a frameshift mutation different from a point mutation?

Chemical Mutagens Chemical damage to DNA can cause 1. Point mutations: When a base is replaced by a different base. * Transitions: purine replaced with a purine, or pyrimidine with a pyrimidine (A-T pair -> G-C pair) - This terminology focuses on the change in the DNA itself, and not the future outcome of the amino acid. * A replaced with G * C replaced with T * Transversions: purine replaced with a pyrimidine, or vice versa. 2. Frameshift mutations: insertion or deletion of a base(s) * Shifts reading frame in translation. * Changes codon with the mutation and all codons after mutation. - Changes the sets of 3 nt (codons) read on the mRNA sequence.

Understand the reaction that DNA polymerase catalyzes. What direction does DNA replication proceed in? Understand the reaction used to synthesize DNA and RNA.

DNA Polymerase Catalyzed Reaction * dNTP's added 1 at a time (deoxynucleoside triphosphate). * Mg2+ ion is also required for DNA polymerases. * Requires a template & 3'-OH to add on to. * Synthesis occurs in the 5' to 3' direction. This is the polymerization reaction catalyzed by DNA polymerase. This (light salmon) is the strand we want to replicate. We always add dNTP's to the 3' hydroxy group. The dNTP's arrive in the active site, again as deoxynucleoside triphosphate. A phosphdiester linkage is created when we have a nucleophilic attack by this (dark salmon) 3' hydroxyl on the innermost phosphorous atom of the deoxynucleoside triphosphate. We see a pyrophosphate is released, that's then hydrolyzed into 2 ions of orthophosphate by pyrophosphatase. That's what's driving polymerization reaction forward. A phosphodiester bond is formed between 3'-OH of DNA strand and 5'PO4 of dNTP. *** Hydrolysis of pyrophosphate to orthophosphate ions drives the rxn forward. Zoom in of the nucleophilic attack by the 3' hydroxyl group on the innermost phosphorous atom of our dNTP. The primer strand must have a free 3' hydroxyl, and as this phosphodiester bridge is formed, a pyrophosphate is released. This is then subsequently hydrolyzed to yield two ions of orthophosphate. This is what's driving the reaction forward. Now, we see sugar-phosphate-sugar, and we'll add another dNTP onto this (light salmon) 3' hydroxyl. Strategy for Replication Another critical protein is primase. The reason we need primase is because on the DNA template, there's no 3' hydroxyl group that can be added onto. This (pointed to with pink arrow) is a template, I'm trying to make a copy, but there's nothing here. So, we need a little section, about 10 nt in length so that we have a free 3' hydroxyl for DNA polymerase to add onto. DNA polymerase is not able to use this template on its own to just start popping in nucleotides. It needs the template and a 3' hydroxyl. So, primase makes this black primer, and then DNA polymerase takes over and continues to synthesize the daughter strand. Overall Direction of DNA Synthesis * But dsDNA is antiparallel and new nt are only added to the 3' OH end. This pic is showing what does NOT happen. Yes, the replication fork opens, yes we'll move in one direction to synthesize both the leading and lagging strands, however, recall that we can only build new strands in the 5' -> 3' direction. So, this (baby pink) strand here won't work. We need to add on to this end (hot pink) not this end (pale pink). DNA Synthesis Occurs Differently on Each Strand This is the correct depiction of what's happening. Here (pale pink) we have our 5' end in grey and then our 3' end (salmon) here. We're producing the antiparallel strand in pink. We have the 5' end and the 3' end (hot pink). So, we're going to keep adding onto this 3' end as we open this fork to the left. Down here (purple), for this parental strand, the one going in the 3' -> 5' direction, we're going to have to synthesize sections of the daughter strand in fragments. The direction of this replication fork continues to open. So, in order to get those fragments, we have to have a looping out. * Leading strand: synthesized continuously. - Begins with a short primer, and then continues on, so you just need the primer once. * Ladding strand: synthesized in fragments. - For each fragment, a primer is synthesized by primase. The okazaki fragments are about 1,000 nt in length, and there will be small gaps, not in nt's but there won't be a phosphodiester bridge between each fragment. That's where DNA ligase comes in. Ligase will create those phosphodiester bonds to seal the fragments together. DNA Replication We're looking at two forks. What's happening at the two forks is the same. Here (light salmon) we have our leading strand. We had a primer, but after those first 10 nt, DNA polymerase took over, and continued to add new complimentary nt to the 3' end. We need our SSBPs keeping the duplex from reforming, we have helicase at the fork to separate the strands. At the lagging strand template (dark salmon), we need primase to make a primer. It's just showing two, but it's actually 10 nt. After that primase lays down the primer, then DNA polymerase can take over. So that's shown here, where the primer was made and now DNA polymerase keeps adding blue nt. It has to stop though because ahead of it, this okazaki fragment has already been synthesized. DNA pol. I will remove the red nt because primase is actually a type of RNA pol. so these nt that make up the primer are ribonucleotides: A,U,C,G with the sugar ribose. This is because, when we're done, we only want DNA, not DNA with some RNA nt. So, those have to be removed. DNA pol. I removes the RNA primer and replaces those nt with DNA nt. * DNA ligase seals fragments together. * DNA pol. I fills in any gaps that exist between the fragments. It's also used for repair and primer removal. * DNA pol. III is for replication. DNA Replication Fork Lets zoom in on the fork and the looping out mechanism. * We won't need to handdraw this. * The overall direction of replication continues in the same direction even though the lagging strand is made in the opposite direction. Notice the blue arrow heading up and the pink arrow heading down (the newly synthesized strand). * DNA pol. III * Helicase separates the two strands. * SSBs keep the two strands separate. * The two yellow portions of DNA polymerase are actually attached to one another. DNA Sliding Clamp * β2 subunit of DNA pol. III. * Keeps DNA pol. III on the template strand. * Allows for high processivity of DNA pol. * Many nt are added without DNA pol. falling off of the template. Has a central cavity through which the DNA template will slide. By clasping the DNA in the ring, the polymerase enzyme is able to move without falling off of the DNA substrate. That ring formation adds to the high processivity of pol. This means the ability to catalyze many consecutive reactions without releasing its substrate. Overview of Replication in Prokaryotes * DnaA recognizes origin of replication, formation of prepriming complex. * DnaB = bacterial helicase, creates the replication fork. - SSB bind ssDNA - DNA gyrase helps unwind the DNA and introduces negative supercoils and topoisomerase I relaxes DNA. * Primase synthesizes the RNA primer. * DNA polymerase III synthesizes DNA, building on RNA primer (main replicative enzyme). * DNA polymerase I removes RNA primer, 5' to 3' exonuclease activity, and fills in the gap. * DNA ligase joins okazaki fragments on lagging strand. Lagging Strand Synthesis Again, we need to make the primer. Once we have that free 3' hydroxyl group, pol. III will continue to add complementary nucleotides. Pol. III detaches once the okazaki fragment ahead of it is reached. Now, pol. I has to replace those RNA sequences from the primer with DNA nt. Then, ligase creates the phosphodiester bridge to link the fragments together.

Be able to recognize the structure of bases found in nucleotides.

Purines Purine Adenine Guanine "Pure as gold" Pyrimidines Cytosine Uracil Thymine "Pyrimidines are 'CUT' in half" We're looking at the five nitrogenous bases. We organize them based on their similarity to purine or pyrimidine. * Purines always base-pair with pyrimidines.

What cofactors are needed by polymerase?

DNA Polymerase requires all four activated precursors: dATP, dGTP, dCTP, and dTTP (building blocks). * Also requires Mg2+ (cofactor).

Differentiate between promoters recognized by each eukaryotic RNA polymerase I, II and III.

Eukaryotic Promoters Let's not forget that promoter sequences aren't just immediately upstream of the start site, but thousands of bp away. Pol. II promoters often include an enhancer that is far away that enhances the binding of Pol. II to the promoter. * DPE: A promoter element that can be within the sequence that will be transcribed. - An enhancer is far away but still critical for RNA pol. II binding. *** Bottom Line: there's a lot of variation in the promoter sequences. Know the difference between downstream (DPE) and upstream (UPE) promoter element. Know enhancers are far away from the txn start site. RNA pol. III promoters, well it depends on the type of RNA that's being made. These consist of conserved sequences that are within the transcribed gene.

Enhancer regions are found in eukaryotic DNA. Be familiar with their function and location.

Pol. II promoters often include an enhancer that is far away that enhances the binding of Pol. II to the promoter. - An enhancer is far away but still critical for RNA pol. II binding. Know enhancers are far away from the txn start site.

What does alternative splicing refer to and what does this provide the cell?

Alternative Splicing * 2 different hormones from 1 gene. * Different cell types produce each. * Each with a different function. We have our pre-mRNA strand/primary transcript, and depending on the cell type we're in, different mRNA products will be produced. Two different hormones are produced, they're coming from the same gene. But different cell types produce different products. These have different functions. We can bring together different combinations of exons in the same gene to give us the mature RNA, giving us different forms of a protein for different tissues and developmental stages and signaling pathways within these different cell types, there are different proteins or transacting splicing factors that will dictate which exons are brought together. Alternative splicing for membrane-bound vs. soluble antibody molecules Alternative splicing will lead to mRNA's that result in different forms of the protein. We have the membrane-bound antibody on the surface of a B cell and the soluble version of that Ab. A. The membrane anchoring portion is actually encoded by its own exon. There are a number of human diseases that are due to defects in alternative splicing.

Know the contributions of the following scientists: Maurice Wilkins, Rosalind Franklin, James Watson, Francis Crick, Meselson, and Stahl.

* Meselson and Stahl's DNA experiments are the experiments that helped us understand that DNA replication is semi-conservative. Watson-Crick DNA Maurice Wilkins and Rosalind Franklin: crystalized DNA and obtained x-ray diffraction data. James Watson and Francis Crick: structural model for DNA. * The structure of the DNA double helix was deduced by Watson and Crick. In order to describe that structure, they depended on data obtained by Rosalind Franklin. It was her x-ray diffraction data performed in his lab (Wilkin's) that helped them infer that DNA must have a helical structure. How do we know that replication is semiconservative? Meselson and Stahl's expts. * Grow E. coli in media with ¹⁵NH4Cl for many generations to make 'heavy DNA'. * Transfer E. coli to ¹⁴N media and determine the density of DNA after each generation. They used two isotopes of Nitrogen: ¹⁵N and ¹⁴N. First, they grew E. coli for many generations using ¹⁵N. That means all the DNA in all of these cells contains ¹⁵N. So, if you break open the cells and extract the DNA and then spin that down in a solution of cesium chloride, you'll see the DNA collecting at one band here (dark salmon, blue band). If we take these E. coli and move them into medium with ¹⁴N only, and we wait one round of replication (20 min assuming they are in their logarithmic phase of growth) all the DNA settled here (light salmon). If we let those E. coli replicate again, still with ¹⁴N as their only isotope of nitrogen avilable, and we spin that down, we'll get a band again here in the middle and then a new band up here (highlighted in pink) at ¹⁴N. As we keep going, we see more and more of the ¹⁴N DNA, but still some of the combination ¹⁴N and ¹⁵N DNA. Over time, most of the DNA contains ¹⁴N. Density gradient equilibrium sedimentation In a gradient of cesium chloride. ¹⁴N and ¹⁵N have a density difference of 1%. * That cesium chloride is in a density gradient. These tubes are spun at very high speeds, in a horizontal orientation, and that leads to the DNA settling at these different points in the tube. In part B, we're looking at a UV absorbance photograph of a centrifuge cell, showing these two distinct bands, and then here densitometric tracing (part C), we can look over time at that absorption photograph. At the zero generation, again all the DNA has ¹⁵N, and as we get closer to that 1st generation, we have the hybrid - the ¹⁴N with ¹⁵N hybrid. This is the red with blue. Then, as we move onto the next generation, we still have the hybrid, but we'll also have the DNA with ¹⁴N incorporated. So, again, the bottom line of this experiment is that DNA replication is semi conservative. It is not conservative, meaning all blue going into a daughter cell, it's not dispersing meaning sections of blue and then sections of newly produced DNA, so let's say this had little sections of red in it, this is NOT what happens. Semi-conservative replication IS what happens.

Distinguish between a nucleoside and a nucleotide. How does this affect the nomenclature for each base?

Nomenclature 1 We see the ribose sugar (highlighted in light salmon). We see there's a hydroxyl group on the 2' carbon, so the sugar must be ribose. If we just had a hydrogen there on the 2' carbon, it would be deoxyribose. The base itself is adenine, but combined with the sugar, we now call it adenosine. * Nucleoside: base + sugar Nomenclature 2 * Nucleotide: nucleoside with ester bond to 1+ phosphoryl groups. What we should know: * She won't ask us to memorize this formal nomenclature, but we should know the differences between nucleoside and nucleotide. * We should also know the carbon numbering on the pentose sugars. * We should also be able to differentiate between ribose and deoxyribose. * We should also be able to recognize the 5 different nitrogenous bases. Nomenclature 3 * Deoxyribose in DNA lacks an -OH at carbon 2. * Deoxyribose is more resistant to hydrolysis without this -OH. *** Know this carbon numbering system *** * The difference between ribose and deoxyribose occurs at the 2' carbon.

Know the function of the 5 different polymerases used in E. coli.

The mechanisms for DNA replication are very similar for prokaryotes and eukaryotes. The enzymes that replicate DNA are called DNA polymerases. There are different subtypes of DNA polymerase in prokaryotes and eukaryotes. 1. Polymerase I: Primer removal and DNA repair. 2. Polymerase II: DNA repair. 3. Polymerase III: Replication. 4. Polymerase IV: DNA repair. 5. Polymerase V: DNA repair.

What is unique about type I introns?

* Single-celled eukaryote, protozoa, Tetrahymena. * Group I introns found in invertebrates. * Has self-splicing RNA, spliced without the aid of any proteins. - The pre-rRNA removes its own introns. * Catalytic RNA called ribozymes. Can remove its own introns without the help of proteins. Ribozyme * Catalytic RNA * How? - RNA is single-stranded and bases can find complementary regions elsewhere in same molecule = unique 3D stx. - Some bases in RNA have functional groups that participate in catalysis. - RNA can hydrogen-bond with other nucleic acid molecules. * 23S rRNA is a ribozyme in the 50S subunit of prokaryotes. - Catalyzes peptide bond formation during translation. Forms the peptidyl transferase center. Recall in ribosomes tRNAs arrive one at a time carrying their uniqe AA and those AA have to be linked to one another. It's the RNA that's in the ribosome that's doing that catalytic work. Self-Splicing mRNA, no Spliceosome Needed (Occurs in group I introns) This rRNA precursor will splice itself using a guanosine cofactor. The guanosine is an energy source, but it's also an attacking group. It becomes incorporated into the RNA that we can see here. It associates with the RNA and attacks that 5' splice site. It makes a phosphodiester linkage here and creates at this end, a 3' hydroxyl group, we call this the upstream exon. Then, this 3' hydroxyl group will attack this 3' splice site to join the two exons and release that pink intron. * Structure of a self-splicing intron from Tetrahymena. * Example of a ribozyme Has a very complex folding pattern, and this structure is critical for its function. Peptidyl Transferase (ribozyme) This pocket is where the 23S rRNA resides, and it is responsible for creating this pink peptide bond that we see here.

Be familiar with the RNA polymerase(s) found in E. coli and in eukaryoes.

Ch 37 - Gene Expression in Eukaryotes Eukaryotes have a nuclear membrane. DNA is housed within the nucleus of a eukaryotic cell whereas DNA in a prokaryotic cell is not enclosed. The product of transcription in a eukaryotic cell is called the primary transcript. That's because it first needs to undergo processing, which includes removal of introns, as well as other modifications. Then, we can call it mRNA, it leaves the nucleus and goes out into the cytoplasm where ribosomes will begin the process of translation. As the protein is being built, we'll call that the nascent protein until it is finished. In prokaryotes, you can actually have simultaneous transcription and translation due to the fact that the product of transcription is already mRNA that does not need processing, and without the nuclear envelope, ribosomes can immediately begin the process of translation. * RNA Polymerase is composed of multiple subunits. * Eukaryotic RNA polymerases are designated with roman numerals while prokaryotic RNA polymerases are designated with greek letters. * Different RNA polymerases for transcribing different types of RNA.

What is the difference between a cis- and trans- acting element regarding transcription? Be familiar with examples of each.

Common cis-acting elements in euk. promoters * Cis-acting elements: DNA sequence that regulates the expression of a gene and these sequences are located on the same molecule of DNA. * TATA box between -30 and -100. - Resembles prokaryotic Pribnow box at -10. - Often paired with an initiator element (-3 -> +5); In doing so, we increase the rate txn. * CAAT and GC boxes found between -40 and -150. - GC boxes found in promoters of constitutively expressed (constantly expressed) genes (not highly regulated). * DPE (downstream core promoter element) at +28 -> +32. DPE: after the +1 txn start site. Trans-acting elements would be transcription factors which are proteins. Here, we're talking about DNA sequences that are critical for transcription. Additional Control Elements Promote Initiation of Txn * Additional transcription factors (trans-acting factors) - Bind upstream sites to increase the rate of txn. - Many are tissue/cell-specific * Enhancer sequences (a cis-acting element) - Can be 1000's of bp upstream of the start site, but are still critical for the initiation of transcription. * In prokaryotes txn factors (CAP) bind RNA pol directly. * In eukaryotes, many proteins bind together (indirectly) = combinatorial control. RNA Pol. II binds with the help of txn factors which can be here near the promoter, but additionally, in order for pol. II to be able to bind, other txn factors also need to bind to DNA. Even though it might seem strange that they're so far away physically, on the DNA strand, mediator proteins like these shown in blue can physically link a txn factor to RNA pol. We call this combinatorial control. Polymerase II binds to the promoter starting there with TFIID, and the TBP at the TATA box. But, enhancer sequences that are far away in the nt sequence bind to the txn factors like these orange activators and then mediator proteins and other proteins that we can call coactivators are all critical for the initiation of txn. Let's not forget that in addition to activator proteins, there are also repressor proteins that will do the exact opposite, and prevent RNA polymerase from binding to the promoter.

Understand the big picture of what happens during DNA recombination.

DNA recombination plays important roles in replication and repair. A. Double-strand breaks can be repaired by recombination. B. DNA recombination is important in a variety of biological processes. Recombination * Recombination: exchange of genetic material between 2 different DNA molecules. * Exchange of pieces of DNA between 2 different DNA molecules to form a new combination of DNA sequences. Recombination relates to repair because breaks in DNA can actually be repaired by recombination. When does Recombination Occur? 1. Replication stalls at an area of DNA damage. 2. Repair of dsDNA breaks. 3. During meiosis, crossing-over between homologous chromosomes. 4. Generate molecular diversity, ex. production of different antibodies. 5. Viruses utilize recombination to insert their genome into the host chromosome (HIV). 6. In the laboratory genetically modified animals, specific genes can be added (knock in mouse) or deleted (knock out mouse). We generate these mice also through recombination events. Bred to have a new/deleted gene as a way to help us understand the gene fxn. Let's say there's a nick in one of the template strands at the replication fork. That would cause the replication fork to collapse. That'll leave a double-stranded break here on one of the daughter double helices. This double-strand break can be repaired through recombination. We can make millions of antibodies to so many different epitopes. That's all happening because recombination. We take a few genes and shuffle small segments to generate the variable region of the heavy and light. Chains and variable regions on the T cell receptor to get an amazing variety of antigen-binding regions. DNA Repair Using Recombination 1. 5' exonuclease binds at site of break and generates single strand DNA (after step 1). 2. Strand invasion: when the strand on the damaged DNA in pink will base-pair with the complementary strand on the undamaged DNA (blue) (after step 2). 3. DNA synthesis. 4. 2nd strand invasion, forming two Holliday junctions (after step 3). 5. Cleave and ligate junctions. After step 4, we see two X structures that get a special name called Holliday junctions, which is the last name of the scientist of discovered them. After step 5, we see two new intact molecules of DNA.

What are the base pairing rules for DNA and RNA, and what interactions hold the 2 bases in opposing DNA strands together?

DNA: A-T, G-C RNA: A-U, G-C * 2 strands held together by hydrogen bonds between 'complimentary bases' that pair according to the 'base-pairing rules'. - A-T & G-C * Base pairing rules allow for the precise transfer of genetic info. 3 hydrogen bonds hold Guanine and Cytosine together. 2 hydrogen bonds hold Adenine and Thymine together. * 1 negative charge at each phosphodiester bond. * Negative charge repels hydroxyl ions that could hydrolyze the phosphodiester bond. Here, we're looking at the sugar phosphate backbone of RNA and DNA. RNA has that 2' hydroxyl group on the sugar (sugar ->phosphate->sugar->phosphate etc.). DNA has the same sugar->phosphate pattern. But, the 2' C has a hydrogen making a deoxyribose sugar. And, the nitrogenous bases for DNA are either A,T,C,G. RNA nitrogenous bases are: A,U,C,G. DNA has an overall negative charge (remember electrophoresis). * Double-helix is stabilized by: 1. Hydrophobic effect, driving bases to the interior of the helix. 2. Van der Waals forces between neighboring bases, an attractive force; base stacking. Rotation of about 36 degrees per base and the bases are stacked on eachother. Recall H-bonds hold the purines and pyrimidines together. RNA Structure * RNA is single-stranded but usually forms complex structures. - Ex. tRNA * Stabilized by Mg2+ * Some RNA molecules have catalytic activity. - Ex. rRNA We can see that base pairing of complementary nucleotides and lots of looping out that occurs with RNA: within the cell, metal ions like magnesium help to stabilize RNA. If we take this nucleotide sequence and examine it more closely, we'll see that it forms this 3D structure. Non-Watson-Crick base pairing in RNA Adenine, guanine and cytosine are all interacting. We'll call this non-watson-crick base pairing in RNA. Of course we know that G & C base pair, but we didn't know that adenine and guanine and adenine and cytosine could be linked together. RNA forms non-watson-crick base pairing, complex structures, and has a variety of functions.

DNA can be denatured by increasing the heat or pH of the solution it is in. How does the absorbance of DNA change when DNA is denatured, and what is meant by Tm?

Denaturing and Annealing DNA * 2 strands of DNA are separated during replication. * Separation can also be achieved with heat, melting or denaturing the DNA. * Each DNA strand has a temperature where it abruptly melts into 2 separate strands, melting temperature (Tm). - Can also be achieved with acid/alkaline conditions. * Once the heat or denaturant is removed, DNA strands will spontaneously reform a double helix, or anneal. * Only DNA strands with a complementary DNA sequence can fully anneal together. DNA can be denatured, and then annealed. Plasmid DNA in a miniprep separates and then reanneals when the pH is returned to neutral. When the two strands are separated, we say the DNA is denatured. Tm can be measured in the spectrophotometer at 260 nm * As DNA denatures, A260 increases. * Absorbance increases due to increasing ssDNA. * Called hyperchromic effect. * Tm = temp. where DNA is 1/2 ssDNA and 1/2 dsDNA. - aka melting temp. We can use the absorbance at 260, which is the wavelength at which double-stranded DNA has the highest absorbance. As we increase the temp, the bases become unstacked and it turns out that they'll actually absorb more UV light. So, at this inflection point, we'll call that the melting temp. So here 71 degrees is the Tm. The fact that ssDNA has a higher absorbance compared to dsDNA, that's called the hyperchromic effect. So, unstacked bases absorb more UV light. Tm: the point where half the DNA is single-stranded and half the DNA is double-stranded. * Tm of DNA increases as the % G+C increases. * G-C held together by 3 hydrogen bonds vs. 2 H-bonds between A-T. Guanines and cytosines are held together by 3 H-bonds, rather than 2. So, you need a higher temperature to separate a strand that has a higher percentage of G's and C's.

Several DNA repair mechanisms found in E. coli were discussed in class; understand what each of the following enzymes/proteins do: MutS, MutL, MutH, DNA photolyase, AlkA, and uvrABC exinuclease.

Detect mismatch - MutS Recruit an endonuclease - MutL MutH is the endonuclease. Endonuclease cleaves DNA near mismatch - MutH. Endo means "within DNA strand". In E. coli uses enzyme AlkA, a glycosylase that cleaves the modified base from the deoxyribose sugar. There's an enzyme called uvrABC exinuclease, exi just means "cut out". So, the dimer and surrounding segments are cut out and then polymerase I will fill in the gap that's shown in pink. And then a phosphodiester bridge created by DNA ligase.

*** NOT ON SG *** Nuclear Horomone Receptors

Gene expression is regulated by hormones. a. Nuclear hormone receptors have similar domain structures. b. Nuclear hormone receptors recruit coactivators and corepressors. Nuclear Hormone Receptors They are in the nucleus, they bind to hormones, and they will regulate gene expression. * 50 members in family. - Upon ligand binding, ligand-receptor complex controls expression by binding to control elements on DNA. * Estrogen receptors belongs to steroid receptor family (not membrane-bound like 7TM family). - Once activated by estrogen, ER translocates to nucleus, binds DNA and controls transcription. * Reaponse to hormones is an important method of controlling gene expression. How can a hormone control whether or not a gene is transcribed? Well, it goes back to proteins that do bind to DNA (NHR). Structure of estradiol receptor. It has four domains. This activation domain, a DNA Binding Domain, a hinge domain, and then a ligand binding domain. * Estradiol: a type of estrogen. Estrogen Receptor * Inactive in absence of estrogen. * Conformational change after estrogen binding allows binding of coactivator protein. * this complex modifies chromatin and transcription machinery to control gene expression. Without binding of the ligand, the coactivator would be unable to bind. Nuclear Hormone Receptor: Ligands bind to and activate these nuclear receptors. In order for them to be active though, we need coregulatory molecules. Extra cellular signals can lead to phosphorylation or even ubiquitination (death tag for protein degradation). *** Bottom line: target genes will be transcribed, mRNA or small RNA, and that leads to protein production and action. The ligand is just one important component in modifying cellular activity. There are many other proteins and signals that are required for function.

How is the compaction of chromatin loosened?

Histone Modification * Histone acetylation decreases affinity of histones for DNA. If DNA is not as tightly bound to histones, it is more open and ready for txn. * Acetylated histones are targets for proteins that open up sites on chromatin and initiate txn. Histone tails are the sites open for modification. When the tails are not acetylated, DNA is very compact and not accessible. When histones are acetylated, the DNA is loosened and now open and ready for txn factors to bind, and to help initiate txn. Histone Acetylation * Attachment of acetyl group to histones loosens histone complex from DNA. * HDAC (Histone Deacetylase) enzymes remove acetyl group. * Histone Acetyltransferase (HAT) adds acetyl group onto histone tails. Histone Acetylation Mechanism 1. Reduces affinity of histones for DNA. * This is what acetylation does. 2. Recruits other components of transcriptional machinery. 3. Initiates remodeling of chromatin. * Chromatin is much more compact when not acetylated. * Know HATS add acetyl groups from acetyl coA. HDACS remove those acetyl groups. Chromatin remodeling * Remodeling engine: Binds specifically to acetylated lysine residues. * The product of step 4 shows the ATP dependent remodeling of chromatin structure, exposing a site for RNA pol. * Final outcome: chromatin remodeling is allowing pol. II to bind, and then begin txn.

Understand the term "transcription bubble".

Open Promoter Complex -> Txn Bubble Its about 17 bp of the double helix that are opened, and then as RNA polymerase moves along, the DNA double helix returns to its original form. Txn - Overview of Elongation * RNA-DNA hybrid about 8 nt long. * Txn bubble moves 17 nm/s = 50 nt added per sec. The transcription bubble (circled in purple), is here in the center. The RNA-DNA hybrid, (the pink and green), is about 8 nt in length. The bubble moves about 17 nm/s, which means 50 nt are added per sec. That's in contrast to DNA polymerase which adds 1000 nt/s. The duplex DNA is unwound at the forward end and rewound at the rear. This RNA-DNA hybrid actually rotates during elongation. * Note Txn bubble. * RNA polymerase also requires Mg2+ as a cofactor. This is another look at that transcription bubble showing us in green the RNA being peeled from the template strand and then extruded from the enzyme.

A point mutation is a change in one base and can be described as a transition or a transversion. What is the difference between these two terms?

Point Mutations * Changing the sequence of 1 base in the DNA may: 1. Alter the amino acid specified by the codon, missense mutation. 2. Create a stop codon, nonsense mutation. 3. The codon may still specify the same amino acid, silent mutation. Silent mutations occur because we have multiple codons encoding the same amino acid. DNA contains the sequence for the corresponding mRNA strand. When that mRNA strand has an error, now the wrong AA will be recruited to the ribosome during translation. We can organize the effects on amino acid as 1,2,3. This terminology focuses on the outcomes in terms of the amino acid sequence. Chemical Mutagens Chemical damage to DNA can cause 1. Point mutations: When a base is replaced by a different base. * Transitions: purine replaced with a purine, or pyrimidine with a pyrimidine (A-T pair -> G-C pair) - This terminology focuses on the change in the DNA itself, and not the future outcome of the amino acid. * A replaced with G * C replaced with T * Transversions: purine replaced with a pyrimidine, or vice versa. 2. Frameshift mutations: insertion or deletion of a base(s) * Shifts reading frame in translation. * Changes codon with the mutation and all codons after mutation. - Changes the sets of 3 nt (codons) read on the mRNA sequence.

Know what enzymes are needed to complete DNA replication and the function of each.

Proteins of Replication 1. Helicase: creates replication fork. 2. Single-stranded binding proteins: stabilize ssDNA. 3. Topoisomerases: alter DNA supercoiling. 4. Primase: adds a RNA primer. 5. DNA polymerase: synthesizes new DNA strands. 6. DNA ligase: creates bonds between DNA fragments. Six critical proteins of replication. The activity of DNA ligase is most critical on that lagging strand, which is synthesized in fragments. Helicase Mechanism * Uses energy of ATP hydrolysis to break hydrogen bonds, thus separating the 2 strands of DNA. * Complex moves like an inch worm up one strand of the DNA. Helicase uses ATP hydrolysis to power strand separation. It has six subunits (green) that form a ring structure. One strand, the one shown in pink, will pass through the hole in the center of the helix, it's bound to two loops (in black) on two adjacent subunits. One of them is bound to ATP while the other to ADP plus Pi. Two of the subunits do not have bound nucleotide. Upon binding of ATP to these two subunits shown in blue, and then release of ADP plus Pi from these two pink subunits, that helicase hexamer will undergo a conformational change and that pulls DNA through the helicase. So, that complex will move like an inch worm up one strand of DNA. Helicase: unwinds parental double helix at replication forks. Single-strand binding protein: binds to and stabilizes single-stranded DNA until it is used as a template. Topoisomerase: relieves overwinding strain ahead of replication forks by breaking, swivleing, and rejoining DNA strands. Primase: synthesizes an RNA primer at 5' end of leading strand and at 5' end of each okazaki fragment of lagging strand. DNA pol III: using parental DNA as a template, synthesizes new DNA strand by adding nt to an RNA primer or a pre-existing DNA strand. DNA pol I: removes RNA nt of primer from 5' end and replaces them with DNA nt added to 3' end of adjacent fragment. DNA ligase: joins okazaki fragments of lagging strand; on leading strand, joins 3' end of DNA that replaces primer to rest of leading strand DNA.

How is the reaction catalyzed by RNA polymerase different from that of DNA polymerase? List the cofactors.

RNA Polymerase Requirements 1. Template (DNA) 2. Activated precursors * Ribonucleoside triphosphates (ATP, GTP, UTP, CTP) 3. Divalent metal cation cofactor * Mg2+ or Mn2+ Rxn of RNA Synthesis is Identical to DNA Synthesis * Difference: the sugar is ribose, not deoxyribose. But, it's a ribonucleoside triphosphate that enters, and this 3' hydroxyl at the terminus of the growing chain, will attack the innermost phosphoryl group of the incoming ribonucleoside triphosphate. Also, as we learned previously, synthesis is driven by hydrolysis of this pyrophosphate. Another important difference is that RNA polymerase does not require a primer. RNA Synthesis * Similar to DNA synthesis: occurs in the 5' -> 3' direction, nucleotides are only added to the 3' OH, requires Mg2+. * Different from DNA synthesis: RNA polymerase does not require a primer, it can start synthesis from scratch. - First nt is a nucleoside triphosphate (G or A). From there, Y and X just means A,U,C, or G. So, any nucleotide is then added to the 3' end. So notice here in pink we're just saying at the 5' end of the mRNA in bacteria, we always find A or G triphosphate.

Transcription factors are important for the initiation of transcription in eukaryotes. Be familiar with TBP, TFIID, and TFIIH.

TFII: Transcription Factors for RNA Pol II * RNA pol II binds the start site for transcription with the help of a set of TFII's. - Transcription factors: A-H * Designated with letter A-H. * Their designation does not indicate the order of binding to the promoter. * Initiation begins with TFIID binding the TATA box. * TBP is a component of TFIID which binds the TATA box. - Binding induces conformational changes in the DNA; the double helix is unwound, widening the minor groove. - Hydrophobic interactions important. The double helix makes extensive contacts with the concave side of TBP using hydrophobic interactions. - 4 Phe residues intercalate between bases in the TATA box. TBP is a TATA box binding protein. TBP binding to the TATA box in DNA Promoter It sits atop a DNA fragment, and in doing so, DNA is significantly unwound and bent. Initiation of Txn in Eukaryotes So, here we've got TFIID which includes the TBP that binds directly to the TATA box. Next, we have TFIIA followed by TFIIB, and then TFIIF, RNA polymerase shown in yellow, TFIIE, and TFIIH will bind the complex, one after the next. TFIIH opens the DNA double helix and phosphorylates the CTD, the carboxy terminal domain. This allows RNA polymerase to leave the promoter and to begin transcription. Phosphorylation of this CTD by TFIIH marks the transition from initiation to elongation. * TFII's along with RNA polymerase II form the Basal Txn Apparatus * More txn factors are required.

Understand the function of telomerase and why it is needed for replication of linear chromosomes. Also, know why telomerase is also said to have reverse transcriptase activity.

Telomeres = Ends of Linear Chromosomes * Protect the ends of the chromosome from degradation. * Loop stabilized by telomere binding proteins. * Telomere is hundreds of repeats of hexamer AGGGTT. * Telomeres synthesized by telomerase. - Unique polymerase that carries its own template of RNA. - Really a reverse transcriptase, copies RNA to make DNA. Our cells deal with these telomeres by: * They circularize, which protects the overhang from being degraded. Also this sequence consists of hundreds of repeats of this hexamer: AGGGTT, and that's no encoding for any gene so it's okay that we're losing a little bit each time because it was just a repeating hexamer. Additionally, our cells carry the enzyme telomerase, which is an amazing polymerase that has its own template of RNA. This enzyme uses the RNA template to make more DNA, so it's just going to keep extending the leading strand so that we don't end up shortening the chromosome too much. It's really a type of reverse transcriptase (using RNA to make DNA). Telomere Formation: We start with the telomere AGGGTT and this pic omits the ezyme itself, but it's just focusing on the RNA portion that sits with the protein itself. So, it uses its own RNA template to synthesize more of those hexamers. Once it gets to "TT" it moves over and begins again so we can start here (pink). AGGGTT. We have a translocation event, and then again AGGGTT, and that just keeps repeating so that the template strand can be elongated and thereby protected. Telomeres * Telomerase acts as template to elongate chromosome. - However, usually not active in most cells. * This is okay because, once the chromosomes get too short in a cell, apoptosis will be induced and that cell will die off (programmed cell death) and now that old cell is removed. * Telomeres shorten as cells divide, and once too short, apoptosis is induced. * In cancer cells, telomerase activity is reactivated. - So a cell doesn't get old, it just keeps dividing. This makes telomerase a potential target for anticancer therapy, and researchers are looking at ways to block expression or its activity as a way to treat cancer, and maybe even prevent cancer. * PIC: At age 65, telomeres are way shorter than they were at birth. * Shorter telomeres = shorter life! - Could we extend life by lengthening telomeres?

Understand the purpose and location (relative to the coding region) of the promoter. What is the consensus sequence of prokaryotic promoters?

The Process of Transcription 1. RNA polymerase binds to the promoter, and DNA unwinds at the beginning of a gene. 2. RNA is synthesized by complementary base pairing of free nucleotides with the nucleotide bases on the template strand of DNA. 3. The site of synthesis moves along DNA; DNA that has been transcribed rewinds. 4. Transcription reaches the terminator. 5. RNA and RNA polymerase are released and the DNA helix re-forms. The promoter is upstream of the gene start site. Transcription begins at the start site and RNA polymerase, shown with the "blue bubble" is just adding complementary nucleotides one at a time to the DNA template.That continues until you get to the end of the gene. At that point, RNA polymerase dissociates from the DNA template and the mRNA strand is translated. Here, we're looking at five sequences from different bacterial promoters. What you're taking note of is the sequence in pink. We call that a consensus sequence. That means, on average, we usually see TATAAT in most prokaryotic promoters. Notice, this is here at -10 because the gene begins at one, at the +1 site. In blue is the start of the gene (+1) * Promoter consensus sequence = -10 consensus sequence. Consensus Sequences in the Promoter * E. coli * consensus means average. * -10 Pribnow box (same as -10 consensus sequence) & -35 consensus sequence (bound by σ factor in RNA polymerase). * Strength of promoter helps regulate Txn frequency. * Strong promoters Txn every 2 seconds; weak promoters every 10 min. Genes that have promoters that are very similar or are identical to these consensus sequences are transcribed very frequently. Whereas genes that have sequences that are very different from these sequences are transcribed at a slower rate. The strongest promoters have sequences that correspond most closely to these consensus sequences. Weak promoters have substitution. We won't need to memorize the sequences in pink, but we do need to know they're called the -35 and -10. We should be able to differentiate the -35 and -10 from promoter elements that we see in eukaryotic genes.

Be familiar with the reactions that take place during RNA splicing. Where does this occur, and what molecules mediate these reactions?

The round-colored balls are actually strands of RNA combined with proteins, and they go by "snRNPs", which stands for "small nuclear ribonucleoprotein particles". There are more than 300 proteins that work together with specialized strands of RNA to form the spliceosome. The spiceosome has a mass of 4.8 megadaltons. We begin with U1 binding to the 5' splice site. U2 binds to this branch point. Then, a preformed complex: U4, U5, U6 will join the assembly, and this is what we call the spiceosome. Interactions between U6 and U2 in pink will displace U1 and U4. Then, the branch site adenosine attacks the 5' splice site (black arrow). At end, we get a lariat intermediate. This is actually a transesterification rxn, and a 2' to 5' phosphodiester bond is formed. 1. U1 binds 5' splice site. 2. U2 binds branch site adenine. - Binding requires ATP. 3. U4, U5, U6 join to form the spliceosome. - Binding requires ATP. 4. 1st transesterification reaction occurs between branch site (A) and 5' splice site. * 2' -> 5' phosphodiester bond formed. Transesterification Reaction of Splicing (not hydrolysis + ligation) Rxn of an alcohol with an ester to form a different alcohol and different ester. The 5' exon is cleaved, and the remaining pre-mRNA molecule forms a lariat. Branch Site "A" Attacks 5' Splice Site A lariat is a loop. The catalytic center of the spliceosome is formed by U2 snRNA, which is shown here in pink, and U6 snRNA that's shown in green. These are bp-ed, that's what the black dots are showing us. U2 is also bp-ed to the branch site of this mRNA precursor. 5. 2nd transesterification rxn. - 3' OH of 5' splice site (exon 1) attacks the phosphodiester bond at the 3' splice site (joining the intron to exon 2). 6. The intron is released in the lariat form. - Splicing is complete. Here's the lariat in grey. Then, in step 5, the 3' hydroxyl of the 5' splice site will attack the phosphodiester bond at the 3' splice site, and in doing so, we're joining the intron to exon 2, and then the intron is released in this lariat form, loop form. Errors in Splicing * Splicing is controlled by: 1. Splice sites in pre-mRNA sequence (cis-acting factors). 2. snRNP's (proteins and snRNA forming the spliceosome; trans-acting factors). Book describes a splicing mutation that will lead to thalassemia. That results in defective hemoglobin, there are many different subtypes but when we say thalassemia, we mean a blood disorder. Pre-mRNA with a mutation in the intron creates a new 5' splice site. So we end up with abnormal mature mRNA. The abnormal mature mRNA has an early stop codon on it, it's going to be degraded. Splicing is controlled by sites in the pre-mRNA. That's what we saw here, we consider that a cis-acting factor. And then snRNP so proteins with their small nuclear RNA's to form the spliceosome as well as other proteins that participate in the splicing process.

Understand what is meant by DNA supercoiling and DNA topoisomers.

Topoisomerases alter DNA supercoiling. Need for Topoisomerases * Creating the replication fork introduces negative supercoils (overwinds). * Supercoils must be removed if helicase is going to continue to separate the DNA strands. We have to prep the double helix for unwinding and topoisomerases do that. So, they will create negative supercoils at the replication fork, so we've unwound a small section, but overwound the section in front of it. DNA has to be locally unwound to expose the single strand templates for replication, and that causes overwinding. The supercoils have to be removed if we're going to have helicase keep separating out these strands. So, topoisomerases will do this. We can separate them into type one and type two. Recall that in bacteria, we use the term DNA gyrase rather than topoisomerases. DNA Gyrase is Essential Some antibiotics block activity of DNA gyrase, which results in death of bacteria.

What transcripts do the eukaryotic RNA polymerases I, II, and III transcribe? Which one is responsible for the synthesis of typical mRNA? How are these RNA polymerases similar and different?

Txn in Eukaryotes * More complex. * Compartmentalized in the nucleus. * 3 different RNA polymerases. * More proteins needed. * Nuclear Hormone Receptors. * Histone Acetylation. Eukaryotes have a Nucleus The sheer presence of the nuclear membrane automatically makes the process of gene expression much different in eukaryotes. The Process of Transcription 1. RNA polymerase binds to the promoter, and DNA unwinds at the beginning of the gene. 2. RNA is synthesized by complementary base pairing of free nucleotides with the nucleotide bases on the template strand of DNA. 3. The site of synthesis moves along DNA; DNA that has been transcribed rewinds. 4. Transcription reaches the terminator. 5. RNA and RNA polymerase are released and the DNA helix re-forms. Transcription begins at the promoter, which is the sequence upstream of the gene itself. The txn start site is at +1. RNA pol. reads the template strand and adds complementary nt, until the end of the gene is reached, and at that point, RNA pol. and the RNA will dissociate. However, this time, we can't call that green strand mRNA. We have to call it the primary transcript because it is definitely not yet mRNA. It needs to be processed. Eukaryotic RNA Polymerases Eukaryotic RNA polymerases are differentiated by the types of transcripts they make and also in their sensitivity to a toxin made by mushroom. That toxin is called alpha - amanitin. Recall, "S": svedburg unit, a measure of the sedimentation rate of those strands in a test tube. * Nucleolus is the area of the nucleus where ribosomal RNA is synthesized, specifically these strands of rRNA (transcripts of pol. I). * mRNA made by RNA pol. II Transcription * DNA is transcribed to make mRNA. * Cell components needed for transcription: - template DNA - RNA polymerase - RNA nucleotides (A, U, C, G) * Types of RNA: mRNA, tRNA, rRNA Eukaryotic RNA Polymerase * Large proteins with different subtypes that have 8-14 subunits. * All are homologous to each other and prokaryotic RNA pol. * RNA pol II (makes the strands that become mRNA) is unique: it's regulated by phosphorylation. - Contains a 220-kd carboxyl-terminal domain (CTD) consensus sequence with multiple repeats of YSPTSPS. - Serine residues "S" phosphorylated (regulation). - Phosphorylated residues bind other factors. * Utilize different promoters to transcribe different RNAs Phosphorylation of the CTD will enhance transcription and recruit other factors required to process the RNA pol. II product.

Understand the advantage of having thymine and not uracil in DNA.

Uracil Repair (in DNA) * Why is T used in DNA and not U? * Cytosines spontaneously deaminate -> U * Any "U" in DNA should be a "C" - CH3 group on T distinguishes it from a deaminated V. - T in DNA instead of U preserves fidelity of DNA. * Deaminated C detected and fixed with base-excision repair, utilizing uracil DNA glycosylase. Summary: uracils in DNA are removed. We have repair mechanisms that can quickly find those deaminated cytosines aka uracils, and remove them. Notice thymine and uracil are very similar in structure, and they're only different by this methyl group. The methyl group on thymine will distinguish thymine from a deaminated cytosine, uracil. Using thymine in DNA will help preserve the fidelity. If uracil was used in DNA, then a correctly placed uracil would be indistinguishable from a uracil that was formed from deamination of cytosine. * Uracils in DNA are deaminated cytosines. * Detected and fixed with base-excision repair, utilizing uracil DNA glycosylase (main enzyme, (1)). * Prevents G-C bp from becoming a A-T bp. - A deaminated cytosine is a uracil, and uracil pairs with adenine. * AP endonuclease (2) * Deoxyribose phosphodiesterase (3) will cleave here (purple) at the backbone and DNA polymerase (4) fills in the gap. DNA ligase (5) seals the gap.

Fill in the blank lines to summarize the trp operon: The regulatory gene encodes a ________________, which is inactive in the absence of tryptophan because it does not bind to the ___________. Thus, ______________ is able to bind to the promoter and transcribe the structural genes E, D, C, B, and A, which encode ____________ for ________________. In the presence of tryptophan, the repressor protein can bind to the ____________ because the shape of the repressor protein has changed now that it is bound to __________. With the repressor protein sitting on the operator, ____________ cannot transcribe E, D, C, B, and A, and thus the enzymes needed to make tryptophan will not be made.

trp Operon We have a regulatory gene which is controlled by this promoter here in green. Here the trp promoter consisting of the green portion plus the operator. The structural genes: E,D,C,B,A, and a polycystronic mRNA strand, and five proteins which ultimately become 3 enzymes needed for tryptophan synthesis. The operon itself is the promoter, the operator, and the structural genes. But, it is under tight control by this repressor protein. Recall in the lac operon, the repressor protein was active. In contrast, the repressor protein that is transcribed and translated here from the regulatory gene is inactive. So, it's not going to bind to the operator, and so the default setting for the trp operon is to be always on. That makes sense of course an E. coli cell needs tryptophan all the time, let's keep this on. In contrast with the lac operon, if glucose is avilable, then keep that off. The default setting for the lac operon is off. Now we're zooming in on the repressor protein. Let's say we take E. coli in the lab and we grow it in a flask of broth that has excess levels of Trp, or it's growing on an agar plate and the agar has extra Trp, or in nature E. coli has entered an area with excess levels of Trp. Why should it keep making more? That Trp binds to the repressor protein and activates it, meaning it can bind to the operator. In doing so, it's going to block RNA polymerase from transcribing E,D,C,B,A so no RNA will be made. Remember, if there's no Trp available, if you don't add it in excess amounts in the lab, or it's not available in the environment, then the repressor protein is inactive. So, it wouldn't bind to the operator and RNA polymerase could transcribe E,D,C,B,A. Closer look at what we mean by 5 genes resulting in 3 enzymes. Again, the bottom line of the Trp operon is to make the enzymes for Trp synthesis, but only make them if you need to. If you don't need to make more, then stop and turn this operon off.