Chapter 28. Biomolecules : Nucleic Acids

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

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)

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

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)

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).

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

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

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

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.

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.

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

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

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

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

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

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

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,

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)

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

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

Codon Assignments of Base Triplets

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

The Structure of tRNA

Processing AminoacyltRNA • 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

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

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

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,3-dideoxyribonucleoside triphosphates (ddNTPs) • Each is modified with a different fluorescent dye molecule

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

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

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

DNA Synthesis: DMT Removal • Removal of the DMT protecting group by treatment with a moderately weak acid

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

DNA Synthesis: Oxidation and Cycling • Phosphite is oxidized to phosphate by I2 • The cycle is repeated until the sequence is complete

DNA Synthesis: Clean-up • All protecting groups are removed and the product is released from the support by treatment with aqueous NH3

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 heat-stable (to survive the unwinding) • Each cycle doubles the amount of material • This is exponential template-driven organic synthesis

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

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

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

What three components make up nucleotides? • disaccharides, heterocyclic aromatic amines, and 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

Select the best name for the molecule below: • guanine monophosphate • guanosine monophosphate • deoxyguanidine monophosphate • deoxyguanosine monophosphate • riboguanidine monophosphate

How many base pairs does it take to complete one turn of DNA? • 2 • 5 • 6 • 10 • It depends on the sequence of bases that make up each turn.

What is the DNA complement to the following sequence?5’-CTGAATCGGA-3’ • 5'-TCCGATTCAG-3' • 5'-AGGCTAAGTC-3' • 5'-GACTTAGCCT-3' • 5'-CTGAATCGGA-3' • 5'-GAATCGGACT-3'

Which of the following is true concerning replication? • Addition of nucleotides to the 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

The picture shown below demonstrates:

The picture shown demonstrates: • 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

Which of the following are produced by transcription? • messenger RNA • transfer RNA • ribosomal RNA • All of these • None of these

In the figure shown, the red DNA strand is the: • sense strand • template strand • coding strand • RNA-like strand • All of these

The codons that make up the genetic code are said to be unambiguous. What does this mean? • 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

Chapter 28. Biomolecules : Nucleic Acids