Chapter 28. Biomolecules: Nucleic Acids Why this Chapter? Last, but not least of the 4 major classes of biomolecules to be introduced To introduce chemical details of DNA sequencing and synthesis 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 2 28.1 Nucleotides and Nucleic Acids 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) 3 Heterocycles in DNA and RNA Adenine, guanine, cytosine and thymine are in DNA RNA contains uracil rather than thymine 4 Nucleotides 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) 5 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). 6 28.2 Base Pairing in DNA: The Watson– Crick Model 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 7 Hydrogen Bonds in DNA The G-C base pair involves three H-bonds The A-T base pair involves two H-bonds 8 The Difference in the Strands 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 9 Grooves 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. 10 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. 11 28.3 Replication of DNA Begins with a partial unwinding of the double helix, exposing the recognition site on the bases Activated forms of the complementary nucleotides (A with T and G with C) associate two new strands begin to grow 12 The Replication Process 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 13 28.4 Transcription of DNA 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 14 Messenger RNA (mRNA) 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 15 Ribosomal RNA (rRNA) 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 16 Transfer RNA (tRNA) 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 17 Transcription Process 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, 18 Transcription of RNA from DNA 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) 19 Mechanism of Transcription 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 resume farther down the chain in another exon, with an intron between that is removed from the mRNA 20 28.5 Translation of RNA: Protein Biosynthesis 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 21 Codon Assignments of Base Triplets 22 The Parts of Transfer RNA There are 61 different tRNAs, one for each of the 61 codons that specifies an amino acid tRNA has 70-100 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 23 The Structure of tRNA 24 Processing Aminoacyl 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 25 28.6 DNA Sequencing 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 26 Analytical Methods The Maxam–Gilbert method uses organic chemistry to cleave phosphate linkages at 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 27 The Sanger Dideoxy and Nucleotides The fragment to be sequenced is combined with: A) A small piece of DNA (primer), whose sequence 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 2,3dideoxyribonucleoside triphosphates (ddNTPs) Each is modified with a different fluorescent dye molecule 28 Dideoxy Method - Analysis 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 29 28.7 DNA Synthesis 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 The 5 OH group on the sugar is protected as its p-dimethoxytrityl (DMT) ether 30 DNA Synthesis: Protection 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 31 DNA Synthesis: DMT Removal Removal of the DMT protecting group by treatment with a moderately weak acid 32 DNA Synthesis: Coupling The polymer-bound (protected) deoxynucleoside reacts with a protected deoxynucleoside containing a phosphoramidite group at its 3 position, catalyzed by tetrazole, a reactive heterocycle 33 DNA Synthesis: Oxidation and Cycling Phosphite is oxidized to phosphate by I2 The cycle is repeated until the sequence is complete 34 DNA Synthesis: Clean-up All protecting groups are removed and the product is released from the support by treatment with aqueous NH3 35 28.8 The Polymerase Chain Reaction 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 heatstable (to survive the unwinding) Each cycle doubles the amount of material This is exponential template-driven organic synthesis 36 PCR: Heating and Reaction The subject DNA is heated (to separate strands) with ◦ Taq polymerase (enyzme) and Mg2+ ◦ Deoxynucleotide triphosphates ◦ Two, oligonucleotide primers, each complementary to the sequence at the end of one of the target DNA segments 37 PCR: Annealing and Growing 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 38 PCR: Growing More Chains 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 See figure 28.9 39 40 What three components make up nucleotides? 1. 2. 3. 4. 5. disaccharides, heterocyclic aromatic amines, and 20% phosphate ions monosaccharides, heterocyclic aromatic amines, and phosphate ions monosaccharides, heterocyclic aliphatic amines, and phosphate ions disaccharides, heterocyclic aliphatic amines, and phosphate ions monosaccharides, heterocyclic aliphatic amines, and sulfate ions 1 20% 2 20% 20% 3 4 20% 5 Select the best name for the molecule below: 20% 1. 2. 3. 4. 5. 20% 20% 20% 3 4 20% guanine monophosphate guanosine monophosphate deoxyguanidine monophosphate deoxyguanosine monophosphate riboguanidine monophosphate 1 2 5 How many base pairs does it take to complete one turn of DNA? 20% 1. 2. 3. 4. 5. 20% 20% 20% 3 4 20% 2 5 6 10 It depends on the sequence of bases that make up each turn. 1 2 5 What is the DNA complement to the following sequence? 5’-CTGAATCGGA-3’ 20% 1. 2. 3. 4. 5. 20% 20% 20% 3 4 20% 5'-TCCGATTCAG-3' 5'-AGGCTAAGTC-3' 5'-GACTTAGCCT-3' 5'-CTGAATCGGA-3' 5'-GAATCGGACT-3' 1 2 5 Which of the following is true concerning replication? 1. 2. 3. 4. 5. Addition of nucleotides to the 20% growing chain takes place in the 3’ to 5’ direction. The process is said to be “conservative.” The process is catalyzed by DNA polymerase. The key step is a nucleophilic attack by the 5’ hydroxyl of deoxyribose upon the γ phosphate of a nucleoside triphosphate. All of these 1 20% 2 20% 20% 3 4 20% 5 The picture shown below demonstrates: The picture shown demonstrates: 20% 1. 2. 3. 4. 5. the replication fork. the semiconservative nature of replication. the antiparallel nature of DNA. how one strand must be made discontinuously while the other can be made continuously. All of these 1 20% 2 20% 20% 3 4 20% 5 Which of the following are produced by transcription? 20% 1. 2. 3. 4. 5. 20% 20% 20% 3 4 20% messenger RNA transfer RNA ribosomal RNA All of these None of these 1 2 5 In the figure shown, the red DNA strand is the: 20% 1. 2. 3. 4. 5. 20% 20% 20% 3 4 20% sense strand template strand coding strand RNA-like strand All of these 1 2 5 The codons that make up the genetic code are said to be unambiguous. What does this mean? 1. 2. 3. 4. 5. All 64 codons are specific for a particular amino acid. Each of the 64 codons codes for a different amino acid. Each of the codons that code for amino acids is specific for only one amino acid. Each of the 64 codons can code for more than one amino acid. None of these 20% 1 20% 2 20% 20% 3 4 20% 5 In the figure below, the part shown in red is the: 20% 1. 2. 3. 4. 5. anticodon. acceptor stem. anticodon loop. aminoacyl group. None of these 1 20% 2 20% 20% 3 4 20% 5 What peptide sequence would be formed by the DNA template strand shown below: 3’-CTA-ACG-GGG-CCC-GCC-5’ 20% 1. 2. 3. 4. 5. 20% 20% 20% 3 4 20% Asp-Pro-Cys-Arg-Gly Asp-Cys-Pro-Gly-Arg Asp-Cys-Pro-Arg-Gly Cys-Pro-Arg-Gly-Arg None of these 1 2 5 What are restriction endonucleases? 1. 2. 3. 4. 5. enzymes that catalyze the hydrolysis of phosphodiester bonds 20% of DNA strands containing a particular base sequence enzymes that randomly catalyze the phosphodiester bonds of DNA strands enzymes that catalyze the disruption of base pairing along an entire DNA strand enzymes that prevent hydrolysis from occurring on a strand of DNA enzymes that prevent nucleic acids from being cleaved 1 20% 2 20% 20% 3 4 20% 5 Which of the following is not required in the chain termination method for DNA sequencing? 20% 1. 2. 3. 4. 5. 20% 20% 20% 3 4 20% ddNTPs dNTPs DNA polymerase radioactive sulfur primer 1 2 5 In synthetic DNA synthesis, what is true about the following reaction? In synthetic DNA synthesis, what is true about the following reaction? 1. This represents the first step of DNA synthesis. 2. This represents the removal of a protection group so the nucleotide can join with another. 3. The removal of the DMT protection group occurs via an SN2 mechanism. 4. This reaction does not occur if the base is thymine. 5. This represents the removal of a protection group so the nucleotide can join with another; and the removal of the DMT protection group occurs via an SN2 mechanism. 20% 1 20% 2 20% 20% 3 4 20% 5 Which of the following is not required for polymerase chain reaction? 20% 1. 2. 3. 4. 5. 20% 20% 20% 3 4 20% RNA polymerase Taq polymerase or another heat-stable polymerase target DNA primers dNTPs 1 2 5 Why was the discovery of Taq polymerase the key to polymerase chain reaction? 1. 2. 3. 4. 5. Taq polymerase is a faster DNA polymerase than that found in mammals. Taq polymerase has a lower error rate than other DNA polymerases. Taq polymerase needs no primer. Taq polymerase does not denature at temperatures of over 90° C, allowing for automated replication. All of these 20% 1 20% 2 20% 20% 3 4 20% 5