John E. McMurry Paul D. Adams University of Arkansas Chapter 28 Biomolecules: Nucleic Acids

 DNA and RNA are chemical carriers of a cell’s genetic information  Coded in a cell’s DNA is the information that determines the nature of the cell, controls cell growth, division  Nucleic acid derivatives are involved as phosphorylating agents in biochemical pathways Nucleic Acids

 Last, but not least of the 4 major classes of biomolecules to be introduced  To introduce chemical details of DNA sequencing and synthesis Why this Chapter?

 Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are the chemical carriers of genetic information  Nucleic acids are biopolymers made of nucleotides, aldopentoses linked to a purine or pyrimidine and a phosphate  RNA is derived from ribose  DNA is from 2-deoxyribose  (the ' is used to refer to positions on the sugar portion of a nucleotide) 28.1 Nucleotides and Nucleic Acids

 Adenine, guanine, cytosine and thymine are in DNA  RNA contains uracil rather than thymine Heterocycles in DNA and RNA

 In DNA and RNA the heterocycle is bonded to C1 of the sugar and the phosphate is bonded to C5 (and connected to 3’ of the next unit) Nucleotides

Nucleotides (Continued)

 Nucleotides join together in DNA and RNA by as phosphate between the 5’-on one nucleotide and the 3 on another  One end of the nucleic acid polymer has a free hydroxyl at C3 (the 3 end), and the other end has a phosphate at C5 (the 5 end). Nucleotides (Continued)

 In 1953 Watson and Crick noted that DNA consists of two polynucleotide strands, running in opposite directions and coiled around each other in a double helix  Strands are held together by hydrogen bonds between specific pairs of bases  Adenine (A) and thymine (T) form strong hydrogen bonds to each other but not to C or G  Guanine (G) and cytosine (C) form strong hydrogen bonds to each other but not to A or T 28.2 Base Pairing in DNA: The Watson–Crick Model

 The G-C base pair involves three H-bonds  The A-T base pair involves two H-bonds Hydrogen Bonds in DNA

 The strands of DNA are complementary because of H-bonding  Whenever a G occurs in one strand, a C occurs opposite it in the other strand  When an A occurs in one strand, a T occurs in the other The Difference in the Strands

 The strands of the DNA double helix create two continuous grooves (major and minor)  The sugar–phosphate backbone runs along the outside of the helix, and the amine bases hydrogen bond to one another on the inside  The major groove is slightly deeper than the minor groove, and both are lined by potential hydrogen bond donors and acceptors. Grooves

Nucleic Acids and Heredity  Processes in the transfer of genetic information:  Replication: identical copies of DNA are made  Transcription: genetic messages are read and carried out of the cell nucleus to the ribosomes, where protein synthesis occurs.  Translation: genetic messages are decoded to make proteins.

 Begins with a partial unwinding of the double helix, exposing the recognition site on the bases  When activated forms of the complementary nucleotides (A with T and G with C) associate, two new strands begin to grow 28.3 Replication of DNA

 Addition takes place 5  3, catalyzed by DNA polymerase  Each nucleotide is joined as a 5-nucleoside triphosphate that adds a nucleotide to the free 3-hydroxyl group of the growing chain The Replication Process

 RNA contains ribose rather than deoxyribose and uracil rather than thymine  There are three major kinds of RNA - each of which serves a specific function  They are much smaller molecules than DNA and are usually single-stranded 28.4 Transcription of DNA

 Its sequence is copied from genetic DNA  It travels to ribsosomes, small granular particles in the cytoplasm of a cell where protein synthesis takes place Messenger RNA (mRNA)

 Ribosomes are a complex of proteins and rRNA  The synthesis of proteins from amino acids and ATP occurs in the ribosome  The rRNA provides both structure and catalysis Ribosomal RNA (rRNA)

 Transports amino acids to the ribosomes where they are joined together to make proteins  There is a specific tRNA for each amino acid  Recognition of the tRNA at the anti-codon communicates which amino acid is attached Transfer RNA (tRNA)

 Several turns of the DNA double helix unwind, exposing the bases of the two strands  Ribonucleotides line up in the proper order by hydrogen bonding to their complementary bases on DNA  Bonds form in the 5  3 direction, Transcription Process

 Only one of the two DNA strands is transcribed into mRNA  The strand that contains the gene is the coding or sense strand  The strand that gets transcribed is the template or antisense strand  The RNA molecule produced during transcription is a copy of the coding strand (with U in place of T) Transcription of RNA from DNA

 DNA contains promoter sites that are 10 to 35 base pairs upstream from the beginning of the coding region and signal the beginning of a gene  There are other base sequences near the end of the gene that signal a stop  Genes are not necessarily continuous, beginning gene in a section of DNA (an exon) and then resuming farther down the chain in another exon, with an intron between that is removed from the mRNA Mechanism of Transcription

 RNA directs biosynthesis of peptides and proteins which is catalyzed by mRNA in ribosomes, where mRNA acts as a template to pass on the genetic information transcribed from DNA  The ribonucleotide sequence in mRNA forms a message that determines the order in which different amino acid residues are to be joined  Codons are sequences of three ribonucleotides that specify a particular amino acid  For example, UUC on mRNA is a codon that directs incorporation of phenylalanine into the growing protein 28.5 Translation of RNA: Protein Biosynthesis

Codon Assignments of Base Triplets

 There are 61 different tRNAs, one for each of the 61 codons that specifies an amino acid  tRNA has ribonucleotides and is bonded to a specific amino acid by an ester linkage through the 3 hydroxyl on ribose at the 3 end of the tRNA  Each tRNA has a segment called an anticodon, a sequence of three ribonucleotides complementary to the codon sequence The Parts of Transfer RNA

The Structure of tRNA

 As each codon on mRNA is read, tRNAs bring amino acids as esters for transfer to the growing peptide  When synthesis of the proper protein is completed, a "stop" codon signals the end and the protein is released from the ribosome Processing Aminoacyl tRNA

 The order of the bases along DNA contains the genetic inheritance.  Determination of the sequence is based on chemical reactions rather than physical analysis  DNA is cleaved at specific sequences by restriction endonucleases  For example, the restriction enzyme AluI cleaves between G and C in the four-base sequence AG-CT Note that the sequence is identical to that of its complement, (3)-TC- GA-(5)  Other restriction enzymes produce other cuts permitting partially overlapping sequences of small pieces to be produced for analysis 28.6 DNA Sequencing

 The Maxam–Gilbert method uses organic chemistry to cleave phosphate linkages with specificity for the adjoining heterocycle  The Sanger dideoxy method uses enzymatic reactions  The Sanger method is now widely used and automated, even in the sequencing of genomes Analytical Methods

 The fragment to be sequenced is combined with: A) A small piece of DNA (primer), having a sequence that is complementary to that on the 3 end of the restriction fragment B) The four 2-deoxyribonucleoside triphosphates (dNTPs)  The solution also contains small amounts of the four 2,3- dideoxyribonucleoside triphosphates (ddNTPs)  Each is modified with a different fluorescent dye molecule The Sanger Dideoxy and Nucleotides

 The product is a mixture of dideoxy-terminated DNA fragments with fluorescent tags  These are separated according to weight by electrophoresis and identified by their specific fluorescence Dideoxy Method - Analysis

 DNA synthesizers use a solid-phase method starting with an attached, protected nucleotide  Subsequent protected nucleotides are added and coupled  Attachment of a protected deoxynucleoside to a polymeric or silicate support as an ester of the 3 –OH group of the deoxynucleoside  Step 1: The 5 –OH group on the sugar is protected as its p- dimethoxytrityl (DMT) ether 28.7 DNA Synthesis

 Step 2: After the final nucleotide has been added, the protecting groups are removed and the synthetic DNA is cleaved from the solid support  The bases are protected from reacting DNA Synthesis: Protection

 Step 2 (Continued): Removal of the DMT protecting group by treatment with a moderately weak acid DNA Synthesis: DMT Removal

 Step 3: The polymer-bound (protected) deoxynucleoside reacts with a protected deoxynucleoside containing a phosphoramidite group at its 3 position, catalyzed by tetrazole, a reactive heterocycle DNA Synthesis: Coupling

 Phosphite is oxidized to phosphate by I 2  The cycle is repeated until the sequence is complete DNA Synthesis- Step 4: Oxidation and Cycling

 All protecting groups are removed and the product is released from the support by treatment with aqueous NH 3 DNA Synthesis- Step 5: Clean-up

 Copies DNA molecules by unwinding the double helix and copying each strand using enzymes  The new double helices are unwound and copied again  The enzyme is selected to be fast, accurate and heat-stable (to survive the unwinding)  Each cycle doubles the amount of material  This is an exponential template-driven organic synthesis 28.8 The Polymerase Chain Reaction (PCR)

 The subject DNA is heated (to separate strands) with  Taq polymerase (enyzme) and Mg 2+  Deoxynucleotide triphosphates  Two oligonucleotide primers, each complementary to the sequence at the end of one of the target DNA segments PCR: Heating and Reaction

 Temperature is reduced to 37 to 50°C, allowing the primers to form H-bonds to their complementary sequence at the end of each target strand PCR: Taq Polymerase  The temperature is then raised to 72°C, and Taq polymerase catalyzes the addition of further nucleotides to the two primed DNA strands PCR: Annealing and Growing

 Repeating the denature–anneal– synthesize cycle a second time yields four DNA copies, a third time yields eight copies, in an exponential series.  PCR has been automated, and 30 or so cycles can be carried out in an hour PCR: Growing More Chains

What amino acid sequence is coded for by the following mRNA base sequence? (5’) CUA-GAC-CGU-UCC-AAG-UGA (3’) Let’s Work a Problem

Answer In order to answer this question effectively, one must refer to Table 28.1, the codon assignments of base triplets. With this reference the amino acid sequence can easily be determined: Leu-Asp-Arg- Ser-Lys-Stop