SURVEY OF BIOCHEMISTRY Nucleic Acids

Which statement is INCORRECT? PRS Question Which statement is INCORRECT? B-DNA is double helical In DNA, two H-bonds form between adenine and thymine In DNA, molar amounts of A+T = molar amounts of G+C DNA strands run antiparallel to each other

Which is not found in DNA? PRS Question Which is not found in DNA? Thymine 2’-OH on pentose 3’-OH on pentose Phosphodiester bond

The link between the adenine base and the ribose sugar occurs: PRS Question The link between the adenine base and the ribose sugar occurs: From N1 of A to the C1’ of ribose From N3 of A to the C1’ of ribose From N7 of A to the C1’ of ribose From N9 of A to the C1’ of ribose

Basics of Nucleic Acid Structure Supercoiling & Topoisomerases RNA Structure Recombinant DNA Technology

Limitations to DNA Flexibility Glycosidic Bond Sugar Pucker Sugar-Phosphate Backbone What is a torsion angle and how does it play a role in biomolecular structure?

Rotational Degrees of Freedom Glycosidic Bond Only 2 permissible conformations of base: syn or anti

Glycosidic Bond Conformations “Anti” conformation is most stable in B-DNA and A-DNA

B-DNA has a C2’-endo conformation… Sugar Pucker B-DNA has a C2’-endo conformation…

A-DNA has a C3’-endo conformation… Sugar Pucker A-DNA has a C3’-endo conformation…

DNA Supercoiling Increasing supercoiling

DNA Supercoiling L = T + W Linking Number (L) Number of times one strand crosses another strand Here the red strand of DNA crosses the blue strand of DNA 10 times L = 10

DNA Supercoiling L = T + W Twist (T) Number of times one strand makes a complete revolution about the helical axis. Here the red strand of DNA makes 10 revolutions about the helical axis T = 10

DNA Supercoiling L = T + W Writhe (W) Number of times a duplex makes a complete revolution about itself. Counterclockwise turns are negative Clockwise turns are positive W = 0

Where do we observe supercoiling of DNA?

Topoisomerases Topoisomerases are enzymes that cleave DNA, thereby relieving supercoiling tension. Type 1A: ss breaks Type 1B: ss breaks Type 2: ds breaks

Types of RNA Messenger RNA - carries genetic information for protein synthesis Transfer RNA - covalently binds amino acids corresponding to its anticodon sequence Ribosomal RNA - RNA molecules that play a role in protein synthesis in conjugate with ribosomal proteins

Transfer RNA Structures Carnegie Mellon U. NIH

Ribosomal RNA Structures Frog 5S rRNA Subunit

Recombinant DNA Technology Goal: Introduce foreign gene into a host organism Reproduction of the gene or gene product Function of the new gene product on the host Players: Gene insert, host, vector used to carry gene into host

Gene Insert Genomic DNA cDNA PCR All of the DNA carrying genetic information for normal functioning of an organism cDNA DNA copied from mRNA PCR DNA that has been copied from a template in a polymerase chain reaction

What is Polymerase Chain Reaction (PCR)? In vitro DNA synthesis Components include: Heat-stable DNA polymerase (Taq polymerase) Two Primers (DNA oligonucleotides) Deoxynucleotides –dATP, dTTP, dCTP, dGTP DNA template Mg++, buffer components, and water

How does PCR work? One PCR Cycle: