Lect 3 bioch2

topoisomerase

*As the DNA unwinds, the area ahead of the replication fork* will actually begin to "supercoil" or "supertwist" To relieve this pressure, special enzymes called DNA Topoisomerases are present. Their job is to clip (nick) one the DNA long strands, let it unwind, and *then re-seal* (ligate) the nick Supercoiling is easily envisioned by thinking about telephone cords that get twisted. To relieve any "knots" that form, unplug the cord, let it relax, and replug it into the telephone Topoisomerases make a transient DNA break during which the topoisomerase enzyme *is covalently attached to the DNA via an active site tyrosine residue* The end result of the reaction is a DNA molecule which is chemically unchanged and covalently closed, but in a different topology

Linear or Cirlcular DNA

*DNA does not exist in a cell as an elongated structure* 􏰀 The linear structure of dsDNA is bound to a complex mixture of proteins including histone proteins and non-histone proteins 􏰀 The combination of dsDNA and proteins define "chromatin"  􏰀 dsDNA can also "cyclize" and form a closed circular molecule 􏰀 Closed circular DNA exists in mitochondria of eukaryotic cells 􏰀 Closed circular DNA also exist in prokaryotes. Most *bacteria* also contain small, circular, extra-chromosomal DNA molecules called plasmids. Plasmids carry genetic information and can replicate independently of chromosomes

dna

*DNA* is a *double*-stranded helix, with the two strands connected by hydrogen bonds. A bases are always paired with Ts, and Cs are always paired with Gs (which is consistent with and accounts for *Chargaff's rule*) The DNA double helix is *anti-parallel* 3. Nucleotides are linked to each other by their phosphate groups, which *bind the 3' end of one sugar* to the 5' end of the next sugar 4. DNA base pairs are connected via *hydrogen bonding*, but the *outer edges* of the nitrogen- containing bases are exposed and *available for potential hydrogen bonding outside* the double helix as well. (These hydrogen bonds provide *easy access to* the DNA for other molecules, including the proteins that play vital roles in the packaging, replication and expression of DNA )

dna and rna

*Deoxy" ribose* has one hydroxyl group removed from the *furanose ring* 􏰅 Note the ribose and deoxyribose ring numbering system *uses a "prime"* (to distinguish its atoms from the nitrogenase base numbering system). The "oxy group is removed from the 2' position in deoxyribose 􏰅 RNA is less stable than DNA because it is *more prone to hydrolysis*. The "extra" hydroxy (electronegative) pulls electrons away from the carbon to the oxygen, making the ribose ring *more susceptible to hydrolysis* by OH groups *The nitrogenous bases will covalently bond to C1' of ribose or deoxyribose* (note: to distinguish the numbered positions on the base from the numbered positions on the ribose sugar, use a ' (prime) mark) 􏰅 Note the location of C3' and C5' - these are the linkage sites that of polymerized nucleic acids

An important cofactor in the synthesis of nucleotides is Folic acid (Tetrahydrofolate) and its 1 carbon "activated form" - N10-Formyl tetrahydrofolate

*N10-Formyl* THF transfers one carbon units at 2 different steps in the synthesis pathway making purine nucleotides 􏰅 *Folic acid derivatives will also transfer *one carbon units in the synthesis of the pyrimidine Thymidine monophosphate (TMP) Some *synthetic inhibitors of purine synthesis* are designed to inhibit the growth of rapidly dividing cells (e.g. cancer cells, microbes) by interfering with folic acid steps, hence depriving the cell of needed nucleotides 􏰅 The "selectivity" of action of these drugs depends on the fact that rapidly dividing cells are in greater need for nucleotides than normal healthy cells and hence are targeted first

IMP

*Nucleotides can be synthesized or recycled. Very little in obtained from the diet* *n important intermediate in the synthesis of purine nucleotides is Inosine monophosphate (IMP). The nitrogenous base of IMP is called Hypoxanthine*

dna sequence

*The 3'􏰁5' phosphodiester bonds link the 3'-hydroxyl group of one nucleotide with the 5'-hydroxyl of an adjacent nucleotide* 􏰀 The resultant long, unbranched chain has polarity.* The 5' end will have a free phosphate attached. The 3' end will have a free hydroxyl group attached* 􏰀* The "distinguishing feature" of different DNA molecules is provided by the different order of the nitrogenous bases (i.e. the sequence) that are attached to the C1' position of the deoxyribose on the long DNA chain 􏰀 By convention, the bases are always written from the 5'end of DNA to the 3'end* 􏰀 The phosphodiester bonds can be cleaved hydrolytically by chemicals, or hydrolyzed enzymatically by family of nucleases Because of the "extra" hydroxy group on C2', RNA is very labile and is easily cleaved by alkali (basic) solutions

Pyrimidine (T, C, U bases) synthesis

*The nitrogenous base is synthesized separate from the ribose sugar, then they are merged 􏰅 Synthesis of pyrimidines starts with aspartic acid, glutamine (a good source of N) and CO2 These factors are also used in the synthesis of purines* *A key intermediate is Carbanoyl phosphate (also an intermediate in the urea cycle) 􏰅 Along the synthesis pathway, the pyrimidine base intermediate is fused with ribose which is offered as PRPP (Phosphoribosylpyrophosphate) 􏰅 The initial end product of pyrimidine nucleotide synthesis is Uridine 5'monophosphate (UMP)*

BAZ form

*There are 3 major structural forms of DNA 􏰆 The B-form (that described by Watson and Crick in 1953) 􏰆 The A form 􏰆 The Z form* The B-form is a right-handed helix with the bases being perpendicular to the helical axes. There are 10 nucleotide residues per complete 360􏰇 turn 􏰀 The A-form is produced by partially dehydrating B-form dsDNA - causing it to compact slightly in a tilted orientation (not exactly perpendicular) - this form is seen in greater amounts in DNA-RNA and RNA-RNA complexes 􏰀 The Z-form is a left-handed helix that has 12 base-pairs/turn. The deoxyribophophate backbone "zig-zags" (hence "Z"-DNA)

topo 1

*These enzymes cut one strand of the double helix* 􏰀 *They have both a nuclease (strand cutting) and a ligase (strand resealing) activity* 􏰀 Topoisomerase I is found in prokaryotes *(bacteria) and in eukaryotes *(yeast, mammalian cells) but each is different and carries out its function in a different manner. By exploiting these differences, *drugs* can be made with specificity, lessening the potential for unwanted side effects

Making Cytosine from ump

*To form the Cytosine pyrimidine (i.e. Cytodine), one first has to make Uridine nucleotide (UMP To convert Uridine to a Cytosine nucleotide and then to a Deoxycytosine nucleotide (to provide substrates for the synthesis of DNA), the C4 keto group must be aminated. First, however, the UMP must be phosphorylated (twice) to UTP (to yank electrons around) 􏰀 Then transfer the amine from Glutamine, which provides the nitrogen 􏰀 Cytosine nucleotide can be used in RNA synthesis or be dehydroxylated for DNA synthesis. To remove the 2' hydroxy, the CTP must first be dephosphorylated to CDP. CDP is a substrate for Ribonucleotide reductase (enzyme specificity helps control this reaction). The phosphate is replaced after the deoxyribose is made*

Nucleotide

A single "unit" of a furanose sugar and a nitrogenous base is called a *"nucleoside"* (No phosphate is part of a nucleoside structure) 􏰅 If the nucleoside has one or more phosphates added, it is referred to as a "nucleotide" or a "nucleoside phosphate" Note the phosphates confer a *negative charge* on the nucleotide. These groups add protons (H+) to the environment, making it *acidic*. Hence, Nucleotides are known as "nucleic acids"

primer and polymerization

An enzyme called* DNA polymerase replicates the DNA* 􏰀 DNA polymerase polymerizes dATP, dGTP, dCTP and dTTP into DNA 􏰀 Replication fidelity occurs in the new DNA strand because of the *specific base pairing* of the incoming deoxyribonucleoside triphosphates (A􏱒T; G 􏱒C) 􏰀 The enzymatic reaction is: 5'NNN-OH-3' + dNTP 􏰁 5'NNNN-OH-3' + PPi *The new strand is always synthesized in the 5' to 3' direction* 􏰀 *A Pyrophosphate (PPi) is released* 􏰀 *A "primer" is a preexisting "starter (short) sequence to help the DNA polymerase "get going"* 􏰀 *The newly synthesized strand is antiparallel to the template strand*

Pie bonding

Another key force holding dsDNA together (in addition to hydrogen bonds) is *hydrophobic pi (􏰃)-stacking* due to the aromatic nature of the nitrogenous bases  Alternating double bonds in ring structures spread the pi electrons over the extent of the molecule 􏰀* The hydrogen bonds between bases are perpendicular to the long axes of dsDNA 􏰀 This aligns the pi bonding character of one base pair with the base pairs in front of it and behind it*

replication fork

As the two DNA strands separate, they form a "V" 􏰀 This is called the *replication fork *- it is the site where active replication occurs 􏰀 *Replication is bidirectional* - replication forks move in opposite directions from the origin, generating a bigger bubble 􏰀 *There are proteins required for DNA strand separation.* These proteins are required for maintaining the separation of the parental strands and for unwinding the double helix ahead of the advancing fork

Denaturing DNA

Because H bonds are so numerous and important in how DNA is held together, If you disrupt the H-bonds, you can separate the two strands of DNA 􏰀 This is easily achieved in the lab by *changing the pH of the solution* (which changes the ionization of the hydrogen bonding groups) or by heating 􏰀 The DNA will "melt" (denature) when sufficient *heat is added*. This is called the "melting temperature" The phosphodiester bonds are maintained (i.e. are NOT broken) by heating. When the DNA is cooled, it will re-form the double strand (there is an inherent biochemical language that is defined by the bases and their specific sequences). This is called *renaturation or reannealing* 􏰀 Because GC base pairs have 3 hydrogen bonds while AT base pairs have 2, dsDNA that has more GC will require more heat to melt. (i.e. it will have a higher Tm)

strucure of dan

DNA is a polymer of deoxyribonucleoside monophosphates covalently linked by 3'􏰁5' phosphodiester bonds (\ 􏰀 DNA exists as a double stranded molecule in which the two strands wind around each other in the form of a double helix 􏰀 There are a few exceptions to DNA being a double stranded molecule - Some viruses have single stranded DNA (ssDNA) [e.g. Chlamydiamicrovirus, Parvoviridae] 􏰀 DNA is found associated with various types of proteins in the nucleus ("nucleoproteins")

packaging of dan

DNA is the repository of genetic information 􏰀 DNA is present in chromosomes in the nucleus and in the mitochondria of eukaryotic cell 􏰀 DNA is complexed with histones and is packaged away in complex tertiary and quaternary structures to form a chromosome The net result is that each DNA molecule is packaged into a mitotic chromosome that is 50,000x shorter than its extended length

initiate with ran primer

DNA polymerases cannot initiate synthesis of a complementary strand of DNA on a totally single stranded DNA template DNA polymerases require a primer -- a short segment of polymerized nucleotides as a kind of "anchoring" structure 􏰀 The primer to start synthesizing DNA is actually a short polymeric segment of RNA 􏰀 The primer binds to the "open strand" of DNA and forms a short double stranded region consisting of an RNA segment base-paired to its complementary DNA segment 􏰀 This brings a free 3'OH group of the last RNA primer nucleotide into the active site of the DNA polymerase. This OH acts as the first acceptor groups of a deoxyribonucleotide brought together by the DNA polymerase Each added nucleotide requires hydrolysis of 2 high energy phosphate bonds NTP􏰁NMP + PPi􏰁Pi + Pi

Another drug that targets nucleic acid synthesis pathways to elicit a favorable pharmacologic effect is Hydroxyurea

Hydroxyurea reduces production of deoxyribonucleotides by inhibiting Ribonucleotide Reductase 􏰅 Hydroxyurea scavenges tyrosyl free radicals that are required for the reductase of this enzyme Hydroxyurea is used in the treatment of cancers like chronic myelogenous leukemia and in treating sickle cell disease

Nucleotide Base Pairing-Driven by and Stabilized by hydrogen bonding

In a dsDNA, the amount of Adenine is always paired with the amount of Thiamine and the amount of Cytosine is always paired with the amount to Guanine 􏰀 The total amount of purines always equals the total amount of pyrimidines A-T base pairs stabilize the dsDNA by forming 2 hydrogen bonds while C-G base pairs stabilize dsDNA by forming 3 hydrogen bonds Hydrogen "bonds to both oxygens and nitrogens in the base structures

Introns and exons

In most instances, there are regions in the mRNA sequence that are not part of the "reading sequence" in making proteins. These must be removed and discarded 􏰀 These "non-coding" sequences are called Introns (intervening sequences). They are "spliced out" of the mRNA sequence using a molecular complex called a "Spliceosome" 􏰀 The regions of RNA that contain the "readable" sequences are termed Exons. Exons from different parts of mRNA are spliced together to make the mature mRNA molecule that is Capped and poly-adenylated Uracil-rich small nuclear RNA (snRNAs) form small nuclear Ribonucleoproteins particles (snRNPs). These structures mediate splicing reactions 􏰀 Mutations at splicing sites can lead to aberent proteins. It is estimated that 15% of all genetic diseases are the result of mutations that affect RNA splicing

PABA and Sulfonamides

Inhibitors - PABA (para-Aminobenzoic acid), Sulfonamides (Sulfa drugs), Methotrexate 􏰅 In bacteria, PABA and Sulfonamides inhibit the synthesis of folic acid (Humans cannot synthesize folic acid so sulfa drugs do not interfere with human purine synthesis) 􏰅 Methotrexate inhibits the reduction of dihydrofolate to tetrahydrofolate. The needed folate coenzyme is reduced in supply, slowing cell growth (i.e. basic of this anti-cancer compound)

Inhibiotors can be toxic

Inhibitors of human purine synthesis are extremely toxic to tissues 􏰅 They will stop nucleotide synthesis in all tissues (not only to the diseased cell or infectious microorganism present) 􏰅 Tissues with rapidly dividing cells will be most affected 􏰄 Fetus 􏰄 Bone marrow 􏰄 Skin 􏰄 Gastrointestinal tract 􏰄 Immune system 􏰄 Hair follicles 􏰅 Individuals taking these medicines can experience adverse effects such as anemia, scaly skin, GI tract disturbances, immunodeficiencies, hair loss

dna proofreading

It is critical that the newly synthesized DNA strand is made without "mistakes" in sequence 􏰀 Misreading the template and putting the wrong nucleotide in the sequence can have deleterious, even lethal consequences 􏰀 If a wrong nucleotide is added to the growing chain, the DNA polymerase enzyme has the capacity to "excise" the wrong nucleotide (at the end of the growing chain) using its inherent Exonuclease activity, and at the same time replace it with the correct nucleotide

nitogenous base numbering system

Just as with proteins, *the "core" nitrogenous bases found in DNA and RNA can be modified* with different side chains, etc into "unusual" bases (??what is this going to do to H-bonding of that base??) 􏰅 These are found in viral DNA and in transfer RNAs or in certain regions of DNA that help to regulate how that site is recognized by nucleic acid binding factors and/or hydrolyzed by nucleic acid cleaving enzymes (like proteases but with nucleic acid targets) Pyrimidines (T, C, U): *Position 1* *is the most electronegative atom in the ring (the Nitrogen that will bond with ribose in a nucleoside)* 􏰅 Purines (A,G): Position 1 is the nitrogen in the pyridine ring furthest removed from that nitrogen in the imidazole ring which will bind with ribose in a nucleoside

Lecture 4

Lecture 4

rna structure

Like DNA, all three types of RNA are unbranched polymeric molecules composed of nucleoside monophosphates linked by phosphodiester bonds 􏰀 RNA differs from DNA in: 􏰆 Having ribose, rather than deoxyribose sugar 􏰆 Having Uracil instead of Thymine 􏰆 Existing as a single strand that folds back on itself into complex structures 􏰀 There are also small "special" molecules of RNA that are found in the nucleus and in the nucleolus that perform special functions (e.g. that mediate splicing reactions) A main role of the nucleolus (a suborganelle within the nucleus) is to synthesize rRNA and assemble ribosomes

pre mrna

Messenger RNA is initially synthesized as a pre-mRNA molecule in the nucleus 􏰀 The first "processing" reaction is to put a "Cap" on the 5' end. The cap is a Methylated Guanosine triphosphate that is placed "backwards" on the 5' terminal ribose sugar (making a 5'􏰁 5' [triphosphate] linkage). This "stabilizes the mRNA and enhances its ability to enter into the protein synthesis machinery 􏰀 Next, add a poly-Adenosine (Poly-A) tail to the 3' end of mRNA. From 40-200 "A"s are added after transcription is finished (post-transcription). The poly A tail also helps stabilize the mRNA and allows it to leave the nucleus, enter into the cytoplasm, and enter into the protein synthesis machinery

histones positve and negative

Most cells have 23 pairs of chromosomes (46 total) 􏰀 Sex cells, the sperm and the egg, have unpaired sets of chromosomes (23 total) Histones, being positively charged, will form strong electrostatic bonds which neutralize both charges and allow for close-packing 􏰀 The metal ion Magnesium, *(Mg++)* also helps neutralize the negative phosphate groups on the DNA double helix

nucleoside analogs

Nucleoside analogs - use a ribose that is missing the key 3' OH needed for chain elongation (2',-3' dideoxyinosine) 􏰀 Use a chemically modified ribose in a nucleoside (e.g. AZT). 􏰀 Use Cytarabine (araC) (an anti-cancer agent) and Vidarabine (ara A) (an anti-viral agent) Note the 2'OH is in the alpha (up) configuration rather than the beta (down) configuration 􏰀 These "drugs" are supplied as nucleosides (no phosphate). They are converted into nucleotides in the body by cellular "salvage" enzymes

Permutations

Number of different possibly permutations of a sequence Y units long where each unit can be X different possibilities is XY 􏰀 For example, a 6-base sequence of DNA has 46 number of possible permutations, or 4096 􏰀 For a 12-base sequence, there are 412 or 16,777,216 possibilities

mrna

Only represents 5% of the RNA in a cell but is the most heterogeneous in terms of size and base sequence - Can vary in length from 500-100,000 nucleotides long 􏰀 Carries the genetic information from the nuclear DNA to the cytosol (where protein synthesis occurs) 􏰀 Sometimes, the message for more than one protein is included in a single mRNA - term this "polycistronic" (often seen in prokaryotes and viruses) 􏰀 mRNA will also contain added 5' "ends" and 3' "ends" that are not turned into proteins. The 3' end is covered with a long sequence of adenine bases (a "poly A tail"). The 5" end has a cap, and often has a leader sequence that helps the synthesized protein sequester into subcellular organelles and fold properly mRNA amplifies genetic information and provides a level of regulation to cellular processes 􏰀 *mRNA is usually synthesized according to the needs of the cell. It is usually rapidly degraded.* 􏰀 By using mRNA, we avoid the possibility that the genetic information stored in DNA will be contaminated

pre rrna

RNA, first made from DNA, is a linear faithful copy of the DNA sequence 􏰀 The primary transcripts of rRNA contain all the information for the subspecies of rRNA. Initially, they are "linked together" (aka Pre-rRNAs) and need to be cleaved into smaller units by enzymes called Ribonucleases Some "end" nucleotide bases are cleaved off by exonucleases (similar to how the ends of proteins are digested by endoproteases) 􏰀 Additional modifications are done at the bases and at the riboses 􏰀 rRNA synthesis and processing occurs in the nucleolus 􏰀 Sugar and base modifications are facilitated by small "extra" pieces of RNA found in the nucleolus (snoRNA) (i.e. Small, nucleolar RNAs) These "guide" the post-transcriptional modifications of RNAs

template

Replication is the process by which DNA is used as a *template* to produce new DNA strands 􏰀 In replication, a chromsome (a pair of DNA molecules wound into a double helix and containing proteins) is replicated into *two daughter chromosomes* 􏰀 Replication occurs *semi-conservatively* (*Conservative replication* would occur if the DNA parent stayed together in one cell and the daughter cell would have a complete new DNA molecule. "Semi- conservative" means each daughter molecule has half the complement of the parent DNA) 􏰀 *Each strand of DNA is used as a template for new strand synthesis* 􏰀 Each daughter chromosome will inherit one of the "copies" of the DNA form the parent molecule 􏰀 *"Fidelity" *means that the information in the DNA is accurately copied

telomerase cont mitotic clocks

Reverse transcriptase enzymes use an RNA template to synthesize DNA (seems backwards) Telomerase has the RNA hexameric sequence 3'~UCCCAA~5' bound in its complex The DNA nucleotides are added to the 3' end of the RNA primer, by DNA polymerase "keeping the length of the telomere sufficiently long to avoid senescence 􏰀 The RNA primer is eventually removed telomeres are "mitotic clocks" in that their length typically is inversely related to the number of times cells have divided 􏱑 If telomerase can be inhibited, you may be able to slow/stop the growth of cancer (let the cells die) 􏱑 If telomerase can be amplified, you may be able to slow the aging process (keep the cells around longer)

Inhibitors and Poisons

Small molecules that target type II topoisomerase are divided into two classes: *Inhibitors and Poisons* 􏰀 Inhibitors include Mitindomide. Such molecules work by inhibiting the ATPase activity needed for Topoisomerase II function by acting as non- competitive inhibitors of ATP 􏰀 Poisons include Quinolone (including Ciprofloxacin). These small molecules target the DNA-protein complex of Topoisomerase II. Some of these molecules lead to increased cleavage, whereas others inhibit re-ligation 􏰀 *Topoisomerase poisons have been extensively used as both anti-cancer and anti-bacterial therapies 􏰀 As antibacterials, ciprofloxacin selectively targets bacterial (and not eukaryotic-like) gyrase. Drug- resistant bacteria often have a point mutation in the gyrase that renders quinolines ineffective*

okazaki fragments

Synthesis of DNA is discontinuous 􏰀 The DNA polymerase will bind to the separated "bubble" areas 􏰀 The DNA polymerase can only "read" the parental nucleotide sequences in the 3'􏰁5' direction so that the new DNA strand is synthesized in the 5'􏰁3' direction 􏰀 But as one strand grows from the left-to-right direction, the opposite strand cannot be as efficiently "read" The leading strand is the one that is in the right direction for DNA polymerase. This strand is synthesized continuously 􏰀 The "lagging" strand is synthesized in short spurts (i.e. "discontinuously"). The short spurts are called Okazaki fragments 􏰀 The Okazaki fragments are eventually ligated to become a single, continuous DNA strand

telemorase

Telomere "ends" are an area of intense biomedical interest 􏰀 As DNA (containing telomeres) gets replicated, the 3' single stranded end is not properly "primed" so faithful replication cannot occur 􏰀 Over time, the single stranded end shortens every time the DNA replicates 􏰀 *Once telomers shorten to some critical length, the cell is no longer able to divide and goes "senescent" - this is a normal process* 􏰀 *In stem cells and germ cells, and in cancer cells, the telomer does NOT shorten and the cells proliferate* 􏰀 *This reactions is controlled by a special enzyme complex called a Telomerase. Telomerase is a "Reverse Transcriptase", adding a short piece of RNA "template" to the single stranded telomere repeat*

telomeres

Telomeres are complexes of non-coding DNA plus protein that are located at the ends of linear chromosomes 􏰀 Telomeres maintain structural integrity of chromosomes by blocking nuclease enzymes from binding/cleaving DNA 􏰀 Telomeres also help enzymes *differentiate* a 3' end of a chromosome from a 3' "nick" in the middle of a DNA molecule that needs repair 􏰀 In humans Telomeres consist of several thousand tandem repeats of a non-coding hexameric sequence -- *AGGGTT* 􏰀 Most of the repeat structure is base paired (i.e. with TCCCAA); however the primary (AGGGTT) strand extends longer as a free "single stranded end" of the chromosome 􏰀 This extended ssDNA segment (100-300 nucleotides long) folds back on itself, forming a "loop" and "hiding" the 3'end away from enzymes that would like to react with it

cell cycle cyclins and cdks

The cell cycle can be divided in two brief periods: 􏱑 Interphase - when the cell grows, accumulates nutrients, and duplicates its DNA 􏱑 Mitosis - when the cell splits into two distinct cells Interphase consists of three distinct phases: G1, S (synthesis), and G2 DNA is duplicated in the S Phase 􏰀 During M phase, there are two tightly coupled processes: 􏱑 Mitosis, where chromosomes are divided between the two daughter cells 􏱑 Cytokinesis, in which the cell's cytoplasm divides forming distinct cells 􏰀 Cells that have temporarily or reversibly stopped dividing are said to have entered a state of quiescence called G0 phase Components of the cell cycle machinery are frequently altered in human cancer 􏰀 Central players are the cyclin-dependent kinases (cdks), which govern the initiation, progression, and completion of cell cycle events 􏰀 The orderly transition between cell cycle phases is controlled by cdk association with cyclins, cdk inhibitors, their state of phosphorylation, and by ubiquitin-mediated proteolysis 􏰀 As malignant cells evolve, this "control of cell cycle activity" is compromised (for e.g. by overexpression of cyclins and loss of expression of cdk inhibitors) 􏰀 A major consequence of this loss of control is "over growth" (i.e. tumors) 􏰀 Because cdks are so fundamental to cell growth, there is great interest in the development of specific kinase inhibitors that would be expected to block cell cycle progression and induce growth arrest.

types of ran

The first step in carrying out the plan encoded for in our DNA is to "copy" the information from DNA to RNA - This process is called Transcription 􏰀 Three main types of RNA are transcribed: 􏰆 Messenger RNA (mRNA) will carry information for the synthesis of proteins Represents* 5-10% *of cellular RNA 􏰆 Ribosomal RNA (rRNA) will form the "factories" where the mRNA will pass through to actually build the encoded protein Represents 10-15% of cellular RNA 􏰆 Transfer RNA (tRNA) will bring amino acid building blocks to the ribosomes to synthesize the encoded protein Represents 75-85% of cellular RNA

RNA Structure

The flow of information from DNA to RNA to protein is known as the "Central Dogma" 􏰀 The Master Plan" for an organism is stored in the DNA 􏰀 It is the RNA, however, that translates the information into working molecules that "carry out" the master plan

compared to prokaryotic

The processes are the same. The enzymes involved are slightly different One notable difference is that eukaryotic DNA is replicated not as "naked" DNA, but as chromatin - a complex mixture of DNA and histones DNA wraps around histone proteins in disc-like structures - forming repeating structural units called Nucleosomes Nucleosomes are spaced at 200 base-pair intervals. Eukaryotic Okazaki fragments are generally 100-200 nucleotides in length (compared to 1000-2000 nucleotides in prokaryotes) The nucleosomes will "slow-down" the movement of DNA polymerase in eukaryotes compared to prokaryotes Another notable difference is that RNA primers are removed by DNA polymerase in prokaryotic cells and by RNase (i.e. RNA hydrolase) in eukaryotic cells

the double helix and drug targeting

The two unbranced chains are coiled around a *common axis* - called the axis of symmetry 􏰀 The 2 chains run* "anti-parallel"* - with the 5'end of one strand being next to the 3'end of the juxtaposed strand 􏰀 The deoxyribophosphodiester groups are "full" of oxygen atoms and strong negative charges from the phosphates - hence they will be very hydrophilic and will want to maintain contact with the aqueous phase 􏰀 *The nitrogenous bases are less "hydrophilic" but are excellent structures to hydrogen bond.* *These parts of the nucleotides will facing inside the double helix 􏰀 The double helix is like a twisted ladder The way the two strands mesh, there will be minor grooves and major grooves* DNA-targeting drugs can alter the bioactivities of DNA groove binding binding , intercalation and covalent addition

histones

There are 5 "families" or classes of histones, designated H1, H2A, H2B, H3 and H4 􏰀 Histones have a lot of arginine and lysine, the 2 predominant positively charged amino acids 2 molecules each of H2A, H2B, H3 and H4, (an octomer) form the core of the nucleosome "bead" 􏰀 The DNA double helix wraps around a single nucleosome ~ 2 times 􏰀 The N terminal ends of these histones can be Acetylated, Methylated or Phosphorylated. These reversible covalent modifications influence how tightly the histones bind to the DNA , and can act as an "on-off' switch to help regulate Replication and Transcription activities of DNA Nucleosomes are packed more tightly - forming a "polynucleosome" (also called a neurofilament) 􏰀 Polynucleosomes assume the shape of a coil, often referred to as a 30 nm fiber The 30 nm chromatin fiber is organized into loops that are archored by a nuclear scaffold containing several proteins 􏰀 Packaging shortens the DNA by >40,000 times Euchromatin is a lightly packed form of chromatin that is rich in gene concentration. It is often (but not always) under active transcription. Euchromatin comprises the most active portion of the genome within the cell nucleus 􏰀 Heterochromatin is a tightly packed chromatin material. It is usually localized to the periphery of the nucleus

Ribosomal RNA (rRNA)

There are distinct species of rRNA in eukaryotic cells (4 sizes) compared to prokaryotic cells (3 sizes) 􏰀 Combine them in different ways to make "big" ribosomal subunits (e.g. 50S; 60S) and "small" ribosomal subunits (30S; 40S) 􏰀 Take advantage of this to design drugs that inhibit prokaryotic protein synthesis and not eukaryotic protein synthesis (selectively killing bacteria) e.g.: Tetracyclin (binds to the 30S prokaryotic rRNA subunit), Erythromycin (binds to the 50S prokaryotic rRNA subunit)

retrovirus

These viruses carry their gene information (genome) in the form of a single stranded RNA molecule (ssRNA) 􏰀 The viral enzyme Reverse Transcriptase takes uses this viral RNA as a template for the synthesis of a complementary DNA segment (that contains viral gene information) 􏰀 This DNA segment gets integrated into the host chromosome where it is replicated, transcribed and translated 􏰀 The pieces to make new viral particles are present, which are put together and are released so the cycle can propagate in new cells

Making the other pyrimidine nucleotie - Thymidine monophosphate - from deoxyuridine monophosphate (dUMP)

Thiamine is the other pyrimidine base found in DNA. It is different from uridine by having an extra methyl (CH3) group added to the pyrimidine ring Where does the methyl group come from? This one carbon transfer comes from the folic acid derivative (N5, N10-methylene-tetrahydrofolate) 􏰀 The form of folic acid used in various reactions is generally dictated by how much "oxidizing power" needs to reside in the coenzyme. "Methylene is more highly oxidized (more energetic) than a "methyl". The formation of* dTMP requires more oxidizing power* Because folic acid derivatives are used in these reactions, *methotrexate* is a drug that can inhibit synthesis of the thymidine. *Another inhibitor is 5-fluorouracil - a potent, irreversible inhibitor of thymidylate synthetase*

rna primers on okazaki

To get the RNA primer synthesized, a specific RNA polymerase called Primase (DnaG) synthesizes a segment of RNA about 10 nucleotides long 􏰀 This primer is complementary to and anti-parallel to the DNA template. (Recall that RNA contains Uracil (not Thymine)) These short RNA sequences are constantly being synthesized at the replication fork on the lagging strand. Only one primer is required for the leading strand 􏰀 When the growing DNA from one "bubble site" meets the RNA primer of a downstream bubble site, the RNA is excised by Exonuclease activity. (An Exonuclease cleaves (hydrolyzes) nucleotides one-by-one from the end of the chain - just like protein exoproteases)

regions transcribed

Transcription is highly selective - some regions of DNA are transcribed, some are not 􏰀 Some regions of DNA will be transcribed into mRNA - others into rRNA or tRNA. Other regions are not transcribed at all 􏰀 The information to carry out specific tasks is stored in DNA defined by how the bases are specifically sequenced 􏰀 The sequence of bases in DNA will define where transcription enzymes will bind to carry out the needed processes 􏰀 RNA polymerases enzymes and a variety of regulatory proteins (transcription factors) will regulate which areas of DNA to transcribe, how much of the DNA to transcribe and where to stop transcription Another important feature is that many RNA transcripts that initially "faithfully" copy complementary bases DNA, will undergo various modifications, including: 􏰆 Addition of RNA "tails" (extra nucleotide chains) 􏰆 Modification of nitrogenase bases 􏰆 Trimming off certain ends 􏰆 Removing certain segments (splicing out) All of these processes work to make functional molecules and to maximize activities

transposons

Transposons are segments of DNA that can be cut out of one place in the DNA sequence, and moved around to another place in the DNA sequence 􏰀 This splicing out 􏱒 moving 􏱒 splicing back in process frequently involves making an RNA intermediate. This RNA intermediate is used as a template by Reverse Transcriptase 􏰀 Transposins that use this RNA intermediate are called Retrotransposons or Retroposons When a (single) microbe develops a resistance to an antibiotic, it can use transposons to transfer this information to neighboring cells, thus more rapidly spreading the resistance trait

dna ligase

When the end of one growing strand runs into the end of a juxtaposed strand, the two segments must be fused" or "ligated" into a single, continuous strand - DNA ligase DNA Ligase - An enzyme that connects two chains into a single chain When the end of one growing strand runs into the end of a juxtaposed strand, the two segments must be fused" or "ligated" into a single, continuous strand - DNA ligase  

Purines--gag Pyrimidines--cit

having 2 ring) The purine ring is a *combination* of a *pyrimidine ring and an imidazole ring (like a histamine amino acid)* The purines Adenine and Guanine are found in DNA and RNA having one ring The pyrimindies Thymine and Cytosine are found in DNA while the pyrimidines* Cytosine and Uracil* are found in RNA Note that these bases differ in terms of the *side chains off of the rings*.* The key side chains are either amine (NH2), Oxy (=O) or methyl (CH3)*. The nitrogens and oxygens give lots of sites for hydrogen bonding (to hold things together)

nucleotides are high energy

in addition to being basic building blocks of DNA and RNA, nucleotides carry high energy bonds that contribute to many reactions of metabolism They serve as *sources of chemical energy *(ATP and GTP) 􏰅 They participate in cellular signaling as secondary messengers (cyclic guanosine monophosphate *(cGMP)* and cyclic adenosine monophosphate *(cAMP)* 􏰅 They are incorporated into important *cofactors* of enzymatic reactions* (Coenzyme A,* FAD, FMN, NAD+ and NADP+) 􏰅 They also serve as carriers of activated *intermediates in the synthesis of some carbohydrates (UDP-glucose 􏰇 glycogen), lipids* (CDP-choline 􏰇 phosphatidylcholine) and conjugated proteins (glycoproteins􏰇UDP-galactose; GDP-mannose) 􏰅 Nucleotides can also be key regulatory compounds that can inhibit or activate key enzymes to drive or otherwise direct metabolic pathways

Exonucleases compared to Endonucleases Hind2 vs. EcoR1

n molecular biology laboratories, specific endonucleases are used to cut DNA at various places so that specific genes and other DNA elements can be spliced into the DNA 􏰀 This allows for recombinant expression of proteins 􏰀 Below are two examples of such enzymes

ribosome

rRNA are found in association with many proteins as components of a complex structure known as the Ribosome 􏰀 Ribosomes are the sites of protein synthesis in a cell 􏰀 rRNA twists and turns back on itself, forming complex structures 􏰀 RNA, just like proteins, have primary, secondary, tertiary and quaternary structures 􏰆 Primary = sequence of nucletides 􏰆 Secondary = helical twisting along a linear strand 􏰆 Tertiary = folding back on itself to form a "clump" which can have various surface projections and internal molecular clefts 􏰆 Quaternary = how RNAs and proteins combine to form a high-order structure with specific function 􏰀 Some rRNAs have enzyme like activities, catalyze reactions. RNA with catalytic activity is called a "Ribozyme". This helps control (i.e. stop) the activity of mRNA

trna modification

tRNAs are also synthesized as long precursors that must be shorted by ribonucleases 􏰀 They also must be further modified to give each a unique identity. Modifications include: 􏰆 Removing sequences form both the 5' and the 3' ends 􏰆 Removing an "intron" (any nucleotide sequence within a gene that is removed by RNA splicing) from the spot where the anti-codon loop will form 􏰆 Adding a -CCA sequence to the 3' end of tRNA (at the spot where the amino acids will be "carried") 􏰆 Reducing certain uracil structures to make dihydrouracil; rearranging the way bases are linked to the riboses, etc

trna

tRNAs are the smallest of the major type of RNA 􏰀 There is at least 1 specific tRNA molecule for each of the 20 amino acids 􏰀 tRNA contain a high amount of unusual bases (e.g. dihydrouracil) and have extensive intrachain base pairing ) (and unique secondary and tertiary structure) 􏰀 Each tRNA is an "adaptor" molecule that carries the proper amino acid to the growing protein chain in the process of building proteins 􏰀 The "anti-codon site" will recognize the message carried in mRNA for a specific amino acid

step1

􏰀 *Step 1 *of the DNA replication process is for the* DNA double helix to "melt" or separate over a small region* 􏰀 The DNA polymerase only uses single stranded DNA (ssDNA) as a template. There are many, many copies of DNA polymerase 􏰀 In eukaryotic cells, the DNA melts at multiple sites along the DNA helix. Generally this occurs at *regions rich in AT base pairs* (why?...how many hydrogen bonds hold together AT base pairs compared to GT base pairs?) 􏰀 Because there are *multiple 'bubble points"* along the DNA double helix, multiple *DNA polymerases can attach and get to work simultaneously, increasing the rate of DNA replication*

top 2

􏰀 *These enzymes bind tightly to the DNA double helix and cut both strands of the DNA* 􏰀 With the strands (transiently) broken *a second segment of DNA passes through the break to relieve pressure* before the broken strands are* re-ligated* 􏰀 *Topoisomerase II is needed by both prokaryotes and eukaryotes* to separate interlocked molecules following chromosomal replication 􏰀 *A type of Topoisomerase II is called DNA Gyrase*. *It is found in bacteria and helps in both relaxing supercoils and in transiently keeping DNA strands apart during transcription* (i.e. the making of complementary RNA) *One way to inhibit Topoisomerase I* is to block the re-ligation site This will prevent DNA from being replicated and stop microorganism (or cancer cell) growth Topo II has been called nature's magician because it literally can move one DNA segment through another 􏰀 The enzyme cleaves a double-stranded DNA, passes a second duplex through the break, and then immediately repairs the broken strands. This enables *Topo II to control the topology of DNA for chromosome segregation and disentanglement* *Some organisms have two isoforms of topoisomerase II : alpha and beta* 􏰀 In *cancers*, topoisomerase II-alpha is highly expressed in highly proliferating cells. In certain cancers, (e.g. peripheral nerve sheath tumors) high expression of its encoded protein is associated with poor patient survival