1. 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

2. 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

3. 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)

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

5. 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)

6. 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).

7. 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

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

9. 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

10. 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.

11. 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.

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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,

19. 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)

20. 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

21. 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

22. Codon Assignments of Base Triplets

23. The Parts of Transfer RNA
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

24. The Structure of tRNA

25. 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

26. 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

27. 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

28. 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

29. 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

30. 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

31. 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

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

33. 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

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

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

36. 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

37. 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

38. 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

39. 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

41. 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

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

43. 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.

44. 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'

45. 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

46. The picture shown below demonstrates:

47. 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

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

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

50. The codons that make up the genetic code are said to be unambiguous
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

51. In the figure below, the part shown in red is the:
anticodon.
acceptor stem.
anticodon loop.
aminoacyl group.
None of these

52. What peptide sequence would be formed by the DNA template strand shown below: 3’-CTA-ACG-GGG-CCC-GCC-5’
Asp-Pro-Cys-Arg-Gly
Asp-Cys-Pro-Gly-Arg
Asp-Cys-Pro-Arg-Gly
Cys-Pro-Arg-Gly-Arg
None of these

53. What are restriction endonucleases?
enzymes that catalyze the hydrolysis of phosphodiester bonds 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

54. Which of the following is not required in the chain termination method for DNA sequencing?
ddNTPs
dNTPs
DNA polymerase
radioactive sulfur
primer

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

56. In synthetic DNA synthesis, what is true about the following reaction?
This represents the first step of DNA synthesis.
This represents the removal of a protection group so the nucleotide can join with another.
The removal of the DMT protection group occurs via an SN2 mechanism.
This reaction does not occur if the base is thymine.
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.

57. Which of the following is not required for polymerase chain reaction?
RNA polymerase
Taq polymerase or another heat-stable polymerase
target DNA
primers
dNTPs

58. Why was the discovery of Taq polymerase the key to polymerase chain reaction?
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