Chapter 28.Chapter 28.Biomolecules: Nucleic AcidsBiomolecules: Nucleic Acids

Why this Chapter?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

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Nucleic acidsNucleic acidsDNA 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

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28.1 Nucleotides and Nucleic Acids28.1 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)

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Heterocycles in DNA and RNAHeterocycles in DNA and RNAAdenine, guanine, cytosine and thymine are in DNARNA contains uracil rather than thymine

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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)

NucleotidesNucleotides

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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).

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28.2 Base Pairing in DNA: The 28.2 Base Pairing in DNA: The Watson–Crick ModelWatson–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

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

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The Difference in the StrandsThe 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

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GroovesGroovesThe 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.

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Nucleic Acids and HeredityNucleic 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.

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28.3 Replication of DNA28.3 Replication of DNABegins with a partial unwinding of the double

helix, exposing the recognition site on the basesActivated forms of the complementary nucleotides

(A with T and G with C) associate two new strands begin to grow

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The Replication ProcessThe Replication ProcessAddition takes place 5 3, catalyzed by DNA

polymeraseEach nucleotide is joined as a 5-nucleoside

triphosphate that adds a nucleotide to the free 3-hydroxyl group of the growing chain

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28.4 Transcription of DNA28.4 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

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Messenger RNA (mRNA)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

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Ribosomal RNA (rRNA)Ribosomal RNA (rRNA) Ribosomes are a complex of proteins and rRNAThe synthesis of proteins from amino acids and

ATP occurs in the ribosomeThe rRNA provides both structure and catalysis

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Transfer RNA (tRNA)Transfer RNA (tRNA)Transports amino acids to the ribosomes where

they are joined together to make proteinsThere is a specific tRNA for each amino acid Recognition of the tRNA at the anti-codon

communicates which amino acid is attached

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Transcription ProcessTranscription ProcessSeveral turns of the DNA double helix unwind,

exposing the bases of the two strandsRibonucleotides line up in the proper order by

hydrogen bonding to their complementary bases on DNA

Bonds form in the 5 3 direction,

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Transcription of RNA from DNATranscription of RNA from DNAOnly one of the two DNA strands is transcribed

into mRNAThe strand that contains the gene is the

coding or sense strandThe strand that gets transcribed is the

template or antisense strandThe RNA molecule produced during

transcription is a copy of the coding strand (with U in place of T)

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Mechanism of TranscriptionMechanism of TranscriptionDNA 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

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28.5 Translation of RNA: Protein 28.5 Translation of RNA: Protein BiosynthesisBiosynthesis

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

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Codon Assignments of Base TripletsCodon Assignments of Base Triplets

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The Parts of Transfer RNAThe 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

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The Structure of tRNAThe Structure of tRNA

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Processing Aminoacyl tRNAProcessing Aminoacyl tRNAAs each codon on mRNA is read, tRNAs bring amino

acids as esters for transfer to the growing peptideWhen synthesis of the proper protein is completed, a

"stop" codon signals the end and the protein is released from the ribosome

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28.6 DNA Sequencing28.6 DNA SequencingThe order of the bases along DNA contains the

genetic inheritance.Determination of the sequence is based on

chemical reactions rather than physical analysisDNA is cleaved at specific sequences by

restriction endonucleasesFor 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

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Analytical MethodsAnalytical MethodsThe 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

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The Sanger Dideoxy and NucleotidesThe 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 fragmentB) 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

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Dideoxy Method - AnalysisDideoxy Method - Analysis The product is a mixture of dideoxy-terminated

DNA fragments with fluorescent tagsThese are separated according to weight by

electrophoresis and identified by their specific fluorescence

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28.7 DNA Synthesis28.7 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

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DNA Synthesis: ProtectionDNA Synthesis: ProtectionAfter 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

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DNA Synthesis: DMT RemovalDNA Synthesis: DMT RemovalRemoval of the DMT protecting group by

treatment with a moderately weak acid

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DNA Synthesis: CouplingDNA Synthesis: CouplingThe polymer-bound (protected) deoxynucleoside

reacts with a protected deoxynucleoside containing a phosphoramidite group at its 3 position, catalyzed by tetrazole, a reactive heterocycle

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DNA Synthesis: Oxidation and DNA Synthesis: Oxidation and CyclingCyclingPhosphite is oxidized to phosphate by I2

The cycle is repeated until the sequence is complete

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DNA Synthesis: Clean-upDNA Synthesis: Clean-upAll protecting groups are removed and the

product is released from the support by treatment with aqueous NH3

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28.8 The Polymerase Chain Reaction28.8 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 materialThis is exponential template-driven organic

synthesis

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PCR: Heating and ReactionPCR: 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

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PCR: Annealing and GrowingPCR: Annealing and GrowingTemperature 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 PolymeraseThe temperature is then raised to

72°C, and Taq polymerase catalyzes the addition of further nucleotides to the two primed DNA strands

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PCR: Growing More ChainsPCR: Growing More ChainsRepeating 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

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What three components make up What three components make up nucleotides?nucleotides?

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1. disaccharides, heterocyclic aromatic amines, and phosphate ions

2. monosaccharides, heterocyclic aromatic amines, and phosphate ions

3. monosaccharides, heterocyclic aliphatic amines, and phosphate ions

4. disaccharides, heterocyclic aliphatic amines, and phosphate ions

5. monosaccharides, heterocyclic aliphatic amines, and sulfate ions

Select the best name for the molecule Select the best name for the molecule below:below:

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1. guanine monophosphate

2. guanosine monophosphate

3. deoxyguanidine monophosphate

4. deoxyguanosine monophosphate

5. riboguanidine monophosphate

How many base pairs does it take How many base pairs does it take to complete one turn of DNA?to complete one turn of DNA?

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1. 22. 53. 64. 105. It depends on the

sequence of bases that make up each turn.

What is the DNA complement to the What is the DNA complement to the following sequence?following sequence?5’-CTGAATCGGA-3’5’-CTGAATCGGA-3’

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1. 5'-TCCGATTCAG-3'

2. 5'-AGGCTAAGTC-3'

3. 5'-GACTTAGCCT-3'

4. 5'-CTGAATCGGA-3'

5. 5'-GAATCGGACT-3'

Which of the following is Which of the following is truetrue concerning replication?concerning replication?

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20% 20% 20%20%20%1. Addition of nucleotides to

the growing chain takes place in the 3’ to 5’ direction.

2. The process is said to be “conservative.”

3. The process is catalyzed by DNA polymerase.

4. The key step is a nucleophilic attack by the 5’ hydroxyl of deoxyribose upon the γ phosphate of a nucleoside triphosphate.

5. All of these

The picture shown below The picture shown below demonstrates: demonstrates:

1. the replication fork.

2. the semiconservative nature of replication.

3. the antiparallel nature of DNA.

4. how one strand must be made discontinuously while the other can be made continuously.

5. All of these

The picture shown demonstrates:The picture shown demonstrates:

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Which of the following are Which of the following are produced by transcription?produced by transcription?

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1. messenger RNA2. transfer RNA3. ribosomal RNA4. All of these5. None of these

In the figure shown, the red DNA In the figure shown, the red DNA strand is the:strand is the:

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1. sense strand2. template strand3. coding strand4. RNA-like strand5. All of these

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

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20% 20% 20%20%20%1. All 64 codons are specific for a particular amino acid.

2. Each of the 64 codons codes for a different amino acid.

3. Each of the codons that code for amino acids is specific for only one amino acid.

4. Each of the 64 codons can code for more than one amino acid.

5. None of these

In the figure below, the part shown in red In the figure below, the part shown in red is the:is the:

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1. anticodon.2. acceptor stem.3. anticodon loop.4. aminoacyl group.5. None of these

What peptide sequence would be formed What peptide sequence would be formed by the DNA template strand shown by the DNA template strand shown below:below:3’-CTA-ACG-GGG-CCC-GCC-5’3’-CTA-ACG-GGG-CCC-GCC-5’

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1. Asp-Pro-Cys-Arg-Gly2. Asp-Cys-Pro-Gly-Arg3. Asp-Cys-Pro-Arg-Gly4. Cys-Pro-Arg-Gly-Arg5. None of these

What are restriction What are restriction endonucleases?endonucleases?

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1. enzymes that catalyze the hydrolysis of phosphodiester bonds of DNA strands containing a particular base sequence

2. enzymes that randomly catalyze the phosphodiester bonds of DNA strands

3. enzymes that catalyze the disruption of base pairing along an entire DNA strand

4. enzymes that prevent hydrolysis from occurring on a strand of DNA

5. enzymes that prevent nucleic acids from being cleaved

Which of the following is Which of the following is not not required in required in the chain termination method for DNA the chain termination method for DNA sequencing?sequencing?

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1. ddNTPs2. dNTPs3. DNA polymerase4. radioactive sulfur5. primer

In synthetic DNA synthesis, what is In synthetic DNA synthesis, what is true about the following reaction? true about the following reaction?

In synthetic DNA synthesis, what is true about the In synthetic DNA synthesis, what is true about the following reaction?following reaction?

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20% 20% 20%20%20%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.

Which of the following is Which of the following is notnot required for polymerase chain required for polymerase chain reaction?reaction?

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1. RNA polymerase2. Taq polymerase or

another heat-stable polymerase

3. target DNA4. primers5. dNTPs

Why was the discovery of Why was the discovery of TaqTaq polymerase the key to polymerase chain polymerase the key to polymerase chain reaction?reaction?

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1. Taq polymerase is a faster DNA polymerase than that found in mammals.

2. Taq polymerase has a lower error rate than other DNA polymerases.

3. Taq polymerase needs no primer.

4. Taq polymerase does not denature at temperatures of over 90° C, allowing for automated replication.

5. All of these