Fundamentals I
DNA Structure
9/15/08 11-12
Chesnokov
Intro slide: Contains all of his information.
Slide 2: Central Dogma of Molecular Biology Information Transfer in Cells
-He will start by reviewing the Central Dogma from Biology. Information encoded in a DNA molecule is transcribed via synthesis of an RNA molecule.
-The sequence of the RNA molecule is "read" and translated into the sequence of amino acids in a protein.
Slide 3: Picture
-It is shown in the illustration here.DNA is the repository of genetic information, andit is propogated through the process of DNA replication and chromosome separation to daughter cells. RNA serves in the expression of this information through the processes of transcription and translation.
Slide 4: Essential Questions
So what I will talk today about is the structure of DNA… then read off slide
-What are the structures of the nucleotides?
-How are nucleotides joined together to form nucleic acids?
-What are the biological functions of nucleotides and nucleic acids?
Slide 5: Outline
So this is an outline of today’s lecture.
What Is the Structure and Chemistry of Nitrogenous Bases?
What Are Nucleosides?
What Is the Structure and Chemistry of Nucleotides?
What Are Nucleic Acids?
What Are the Different Classes of Nucleic Acids?
So I’m going to talk about what kind of secondary structure DNA can adopt.
We’ll talk about methods to study secondary structure.
We’ll talk about what tertiary structure of DNA is and the structure of eukaryotic structures.
Slide 6: Structure and Chemistry of Nitrogenous Bases
The bases of nucleotides and nucleic acids are derivatives of either pyrimidine or purine.
There are three Pyrimidines in nucleic acids
–Cytosine (DNA, RNA)
–Uracil (RNA)
–Thymine (DNA)
–
There are two Purines in nucleic acids
–Adenine (DNA, RNA)
–Guanine (DNA, RNA)
Slide 7: Ring systems
On the left is the pyrimidine ring system (six-membered heterocylic aromatic rings with two nitrogen atoms); by convention, atoms are numbered as indicated. On the right is the purine ring system consists of two rings (pyrimidine and imidazole), nine atoms numbered as shown here. Both are relatively insoluble in water due to aromatic character.
Slide 8: Pyrimidine rings
These are common pyrimidine bases – cytosine (DNA and RNA), uracil -only in RNA, and thymine-only in DNA. And the full name for cytosine, for example, would be 2-oxy-4-aminopyrimidine and so on. This is an idea for the nomenclature.
Slide 9: Purine rings
The common purine bases —adenine and guanine and both are found in both DNA and RNA. For example, adenine is called 6-amino purine. Guanine is called 2-amino-6-oxypurine.
Slide 10: What Are Nucleosides?
(He read the slide.)
-When a base is linked to a sugar it’s called a nucleoside.
-The sugars are pentoses
-D-ribose (in RNA)
-2-deoxy-D-ribose (in DNA)
-The difference those two is 2'-OH vs 2'-H of the pentose ring
-This difference affects secondary structure and stability of nucleic acids because DNA’s are significantly more stable which makes sense because they are the depository of genetic material. RNA is degraded after its function is finished.
Slide 11: Picture
Pentose forms a sugar in the nucleoside. It is a five membered ring is known as furanose. Furanose structures—ribose and deoxyribose are presented. The presence of a hydroxyl group at the 2-position has dramatic effect on secondary structures available to DNA and RNA as well as their susceptibilities to hydrolysis. DNA needs to be more stable.
Slide 12: continued …
-Base in nucleosides is linked to a sugar via a glycosidic bond
-1’C of sugar links to 9 N of purine or to the 1 N of pyrimidine base
-Nucleosides named by adding –idine (ur-idine) to the root name of a pyrimidine or –osine (aden-osine) to the root name of a purine (for example uridine or odine-not very clearly stated)
-Sugars make nucleosides more water-soluble than free bases
Slide 13:
-This is an example of a glycosidic bond.
-b-Glycosidic bonds link nitrogenous bases and sugars to form nucleosides.
1’C links to 9 N of purine and to the 1 N of pyrimidine base.
Slide 14: Common Ribonucleotides
The common ribonucleosides—cytidine, uridine, adenosine, and guanosine. Also shown, is the unusual nucleoside inosine (uncommon nucleoside but is found in various organisms) is drawn.
Slide 15: Structure and Chemistry of Nucleotides
Nucleotides or Nucleoside phosphatesresult when phosphoric acid is esterified to a sugar-OH group of nucleoside (at C-5)
-Important to know the nomenclature
-Most nucleotides are ribonucleotides
-Nucleotides have acidic properties
-Nucleic acids, which are polymers of nucleosides derive their names from the acidity of phosphate groups.
Slide 16: Structures of the four common ribonucleotides
-This is illustrated in this slide.
-Structures of the four common ribonucleotides —AMP, GMP, CMP, and UMP—together with their two sets of full names, for example, adenosine 5'-monophosphate and adenylic acid. Also shown is the nucleoside 3'-AMP (uncommon, product of hydrolysis).
Slide 17: Functions of Nucleotides
-What are functions of nucleosides? NTP’s are the bases for DNA.
-Nucleoside 5'-triphosphates are carriers of energy. Energy is stored in phosphoric bonds.
-Nitrogenous bases serve as recognition units or information symbol but not involved in the biochemistry of metabolism
These are the most common functions for the bases:
-ATP is central to energy metabolism (energy currency)
-GTP drives protein synthesis
-CTP drives lipid synthesis
-UTP drives carbohydrate metabolism
There are also cyclic nucleotides that are found in the cell and they are important signal molecules and regulators of cellular metabolism and reproduction.
Slide 18: Phosphoryl and pyrophosphoryl group transfer
This is an example of phosphoryl and pyrophosphoryl group transfer, the major biochemical reactions of nucleotides. Phosphoric bonds are prime source of chemical energy to do biological work (ATP, GTP, CTP and UTP, also deoxy- counterparts).
Slide 19: Cyclic nucleotides are cyclic phosphodiesters.
-Structures of the cyclic nucleotides are essentially phosphorous diesters, cAMP and cGMP. Phosphoric acid is esterified to two of the available ribose hydroxyl groups at positions 5 and 3.
-Important! They are regulators of cellular metabolism and are found in all cells.
Slide 20: What are Nucleic Acids? Polynucleotides!(the short answer)
-They are linear polymers of nucleotides linked 3' to 5' by phosphodiester bridges
-Ribonucleic acid and Deoxyribonucleic acid
-Know the shorthand notations (very important to know)
-Sequence is always read 5' to 3' direction
-In terms of genetic information, this corresponds to "N to C" in proteins
Slide 21:Here is the sequence of RNA and DNA and 3'-5' phosphodiester bridges link nucleotides together to form polynucleotide chains. They look similar for RNA and DNA with the difference of a hydroxyl group.
Slides 22 and 23:Shorthand notations for polynucleotide structures.
Furanoses are represented by vertical lines; phosphodiesters are represented by diagonal slashes in this shorthand notation for nucleic acid structures. Bases serve as distinctive side chains and give the polymer its unique identity. Sometimes for shorthand notations only the letters are used for the nucleic acids.
Slide 24: What Are the Different Classes of Nucleic Acids?
-DNA - one type, one purpose (depository for genetic information)
-RNA - 3 (or 4) types, 3 (or 4) purposes
–ribosomal RNA - the basis of structure and function of ribosomes
–messenger RNA - carries the message which is transcribed through the forms of proteins
–transfer RNA - carries the amino acids
–Small nuclear RNA
–Small non-coding RNAs which serve regulatory functions in cells
Slide 25: RNA and DNA differences?
Why does DNA contain thymine?
-Cytosine spontaneously deaminates to form uracil (C-G pair could result in U-A)
-Repair enzymes recognize these "mutations" and replace these U’s with ‘Cs
-But how would the repair enzymes distinguish natural U from mutant U
-Nature solves this dilemma by using thymine (5-methyl-U) in place of uracil
Slide 26:Deamination of cytosine forms uracil. He shows the thymine, which has the additional methyl group at the position 5 and allows it to be recognized as a base that doesn’t need to be replaced. This is important for DNA because it serves as a repository of genetic information but RNA does its function and is quickly destroyed. So, if there is a mutation with one of the mRNA’s it wouldn’t be noticed because there are many many that are formed and perform their function.
Slide 27:The 5-methyl group on thymine labels it as a special kind of uracil.
Slide 28:Why is DNA 2'-deoxy and RNA is not?
-Vicinal -OH groups (2' and 3') in RNA make it more susceptible to hydrolysis
-DNA, lacking 2'-OH is more stable
-This makes sense - the genetic material must be preserved
-RNA is designed to be used and then broken down
Slide 29:How Do Scientist Determine the Primary Structure of Nucleic Acids?
He summarized it for our information, but he usually likes to spend more time on it.
Sequencing Nucleic Acids
-The most common is the chain termination method (dideoxy method), developed by F. Sanger
-Base-specific chemical cleavage, developed by Maxam and Gilbert
-Both use autoradiography - X-ray film develops in response to presence of radioactive isotopes in nucleic acid molecules
Slide 30: DNA Replication
-Chain termination method is based on biochemistry of DNA replication
-Each strand of the double-helical DNA molecule must be copied in complementary fashion by DNA polymerase
-Each strand can serve as a template for copying
-DNA polymerase requires template and primer
-Primer: an oligonucleotide that pairs with the end of the template molecule to form dsDNA
-DNA polymerases add nucleotides in 5'-3' direction
Slide 31: This is a reaction used for the chain termination method. DNA polymerase copies single stranded DNA in vitro in the presence of the four deoxynucleotide monomers. A double-stranded region of DNA must be artificially generated by adding a primer(short strand), an oligonucleotide capable of forming a short stretch of double stranded DNA by base pairing with the single stranded DNA. The primer must have a free 3'-OH end from which the new polynucleotide chain can grow as the first residue is added in the initial step of the polymerization process.
Slide 32: Chain Termination Method
(He read the slide)
Based on DNA polymerase reaction
-Run four separate reactions
-Each reaction mixture contains dATP, dGTP, dCTP and dTTP, one of which is P-32-labelled
-Each reaction also contains a small amount of one dideoxynucleotide: either ddATP, ddGTP, ddCTP or ddTTP
Slide 33: Chain Termination Method
-Most of the time, the polymerase uses normal nucleotides and DNA molecules grow normally
-Occasionally, the polymerase uses a dideoxynucleotide, which adds to the chain and then prevents further growth in that molecule
-Random insertion of dd-nucleotides leaves (optimally) at least a few chains terminated at every occurrence of a given nucleotide
Slide 34: Chain Termination Method
-Run each reaction mixture on electrophoresis gel
-Short fragments go to bottom, long fragments on top
-Read the "sequence" from bottom of gel to top
-Convert this "sequence" to the complementary sequence
Slide 35: Reaction
The chain termination or dideoxy method of DNA sequencing. (a) DNA polymerase reaction. (b) Structure of dideoxynucleotide. (c) Four reaction mixtures with nucleoside triphosphates plus one dideoxynucleoside triphosphate (either A,G,C or T). (d) Electrophoretogram. Note that the nucleotide sequence as read from the bottom to the top of the gel is the order of nucleotide addition carried out by DNA polymerase. Bends are determined by autoradiography.
Slide 36: Chemical Cleavage Method
Not used as frequently as Sanger's
-Start with ssDNA labelled with P-32 at one end
-Strand is cleaved by chemical reagents
-Assumption is that strands of all possible lengths will be produced, each cleaved at just one of the occurrences of a given base.
-Fragments are electrophoresed and sequence is read
Slide 37:
Here you can see a series of gel containing the four lanes, A,T,G, and C. Sequences can be read from bottom to top. We don’t really around (?) these gels in the lab anymore because there are so many core facilities, but when I was a graduate student we used to do it all the time. It is still a useful method for other techniques, like primer extension and other techniques.
Slide 38: DNA Structure
summary
The fundamental structure of DNA is a Double Helix Stabilized by hydrogen bonds!
-DNA consists of two polynucleotide strands wound together to form DNA double helix
-Strands run in opposite direction (antiparallel)
-Two strands are held together through interchain hydrogen bonds
-These H bonds pair the bases of nucleotides in one chain to complementary bases in the other, called base pairing.
Slide 39: The DNA Double Helix
-Erwin Chargaff had the pairing data, but didn't understand its implications. Chargaff rule – the number of purine residues equals the number of pyrimidine residues in all organisms (A=T, G=C).
-Rosalind Franklin's X-ray fiber diffraction data was crucial in discovering DNA because she showed it was a Helix!
-Watson-Crick (received a Nobel Prize) model of the DNA double helix.
Slide 40: Here we see a DNA model. (a) Double-stranded DNA as an imaginary ladderlike structure. (b) A simple right-handed twist converts the ladder to a helix.
Slide 41:
A model of DNA double helix.
The nucleotides are linked covalently by phoshodiester bonds through the
3’-hydroxil (-OH) group of one sugar and the 5’-phosphate (P) of the next.
Two DNA strands are held togetherby hydrogen bonds between the paired bases. Two hydrogen bonds form between A and T, while three form between G and C. The bases can pair in this way only if the two polynucleotide chains that contain them are antiparallel to each other.
The coiling of the two strands around each other creates two grooves (major and minor) in the double helix.
Consequences – each strand of DNA contains a sequence of nucleotides that are exactly complementary tothe sequence of its partner strand.
Slide 42: This demonstrates how grooves form. The bases in a base pair are not directly across the helix axis from one another but rather are slightly displaced. This displacement, and the relative orientation of the glycosidic bonds linking the bases to the sugar–phosphate backbone, leads to differently sized grooves in the cylindrical column created by the double helix, the major groove and the minor groove, each coursing along its length. The major groove is large enough to accommodate alpha-helix of a protein and many transcription factors and regulatory proteins, there are actually proteins that can recognize the major or minor grooves of DNA.
Slide 43:Comparison of A, B, Z DNA
A: right-handed, short and broad, 2.3 Å, 11 bp per turn (dehydrated DNA, probably does not exist in vivo)
B: right-handed, longer, thinner, 3.32 Å, 10 bp per turn, (most common)
Z: left-handed, longest, thinnest, 3.8 Å, 12 bp per turn (G-C rich regions)
Slide 44:Comparison of the A-, B-, and Z-forms of the DNA double helix. The distance required to complete one helical turn is shorter in A-DNA than it is in B-DNA. The alternating pyrimidine–purine sequence of Z-DNA is the key to the “left-handedness” of this helix. Remember that B-structure is the most common found in organisms and Z-structure is only in the G-C regions.
Slide 45:Can the Secondary Structure of DNA Be Denatured and Renatured?
Important for Study of Genome complexity
Scientists found that DNA can be denatured and renatured.
-When DNA is heated to 80+ degrees Celsius,its UV absorbance increases by 30-40% (because of the aromatic rings)
-This hyperchromic shift reflects the unwinding of the DNA double helix
-Stacked base pairs in native DNA absorb less light
-When temperature is lowered, the absorbance drops, reflecting the re-establishment of stacking
Slide 46:
When T is increased, two DNA strands can be separated, but when T is gradually decreased, two strands can find eachother through the process of nucleation and aligning sequences onto complimentary strands. This is called zippering. But this process depends on the complexity of DNA. If you have a simple polyA with a polyT they can reanneal quickly. However, if you have a complicated strand from a mammalian species it might take hours for DNA to find eachother, hours for complementary strands to anneal.
Slide 47:If you have a simple DNA sequence poly U poly A, then it can reanneal quickly. However, if you have Kauhof (?) repetitive section of DNA it may take hours for those strands to find eachother. This method was popular, and many scientists found many satellite DNA’s, which are repetitive, in many species. It was a great way to study the complexity of genomes.
Slide 48: Tertiary Structure of DNA
-Essentially what it is, the DNA helix can adopt a superhelical or supercoiled state in which DNA can be either under- or overwound. The enzymes that help with this are called topoisomerases or gyrases can introduce or remove supercoils
-In duplex DNA, ten bp per turn of helix
-Circular DNA sometimes has more or less than 10 bp per turn - a supercoiled state, underwound (-) or overwound (+).