Biochemistry Exam 2: Chapter 9 (Nucleic Acids: How Structure Conveys Information)
Consider the following in light of the concept of levels of structure (primary, secondary, tertiary, quaternary) as defined for proteins. (a) What level is shown by double-stranded DNA? (b) What level is shown by tRNA? (c) What level is shown by mRNA?
(a) Double-stranded DNA is usually thought of as having secondary structure, unless we consider its supercoiling (tertiary) or association with proteins (quaternary) (b) tRNA is a tertiary structure with many folds and twists in three dimensions (c) mRNA is usually considered a primary structure, as it has little other structure.
2. Recall What is the structural difference between thymine and uracil? 3. Recall What is the structural difference between adenine and hypoxanthine?
2. Thymine has a methyl group attached to carbon 5; uracil does not. 3. In adenine, carbon 6 has an amino group attached; in hypoxanthine, carbon 6 is a carbonyl group
47. Reflect and Apply Would you expect tRNA or mRNA to be more extensively hydrogen bonded? Why? 48. Reflect and Apply The structures of tRNAs contain several unusual bases in addition to the typical four. Suggest a function for the un-usual bases. 49. Reflect and Apply Would you expect mRNA or rRNA to be degraded more quickly in the cell? Why?
47. More extensive hydrogen bonding occurs in tRNA than in mRNA. The folded structure of tRNA, which determines its binding to ribosomes in the course of protein synthesis, depends on its hydrogen-bonded arrangement of atoms. The coding se- quences of mRNA must be accessible to direct the order of ami- no acids in proteins and should not be rendered inaccessible by hydrogen bonding. 48. They prevent intramolecular hydrogen bonding (which occurs in tRNA via the usual A-U and C-G associations), thus permit- ting loops that are critical for function, the most important be- ing the anticodon loop. 49. Turnover of mRNA should be rapid to ensure that the cell can respond quickly when specific proteins are needed. Ribosomal subunits, including their rRNA component, can be recycled for many rounds of protein synthesis. As a result, mRNA is degraded more rapidly than rRNA.
8. Reflect and Apply (a) Is it biologically advantageous that DNA is stable? Why or why not? (b) Is it biologically advantageous that RNA is unstable? Why or why not?
8. (a) Definitely yes! If there is anything that you don't want falling apart, it's your storehouse of genetic instructions. (Compare the effectiveness of a computer if all the *.exe files were de- leted.) (b) In the case of messenger RNA, yes. The mRNA is the trans- mitter of information for protein synthesis, but it is need- ed only as long as a particular protein is needed. If it were long-lived, the protein would continue to be synthesized even when not needed; this would waste energy and could cause more direct detrimental effects. Thus, most mRNAs are short-lived (minutes); if more protein is needed, more mRNA is made.
31. Reflect and Apply Would you expect to find adenine-guanine or cytosine-thymine base pairs in DNA? Why?
Adenine-guanine base pairs occupy more space than is available in the interior of the double helix, whereas cytosine-thymine base pairs are too small to span the distance between the sites to which complementary bases are bonded. One would not nor- mally expect to find such base pairs in DNA.
24. Recall What is the difference between B-DNA and Z-DNA?
B-DNA is a right-handed helix with specified dimensions (10 base pairs per turn, significant differences between major and minor groove, etc.). Z-DNA is a left-handed double helix with different dimensions (12 base pairs per turn, similar major and minor grooves, etc.).
39. Reflect and Apply A technology called PCR is used for replicating large quantities of DNA in forensic science (Chapter 13). With this technique, DNA is separated by heating with an automated system. Why is information about the DNA sequence needed to use this technique?
Because any system involving replication of DNA by DNA poly- merases must have a primer to start the reaction, the primer can be RNA or DNA, but it must bind to the template strand being read. Thus, enough of the sequence must be known to create the correct primer.
46. Recall What is RNA interference?
RNA interference is the process by which small RNAs prevent the expression of genes.
G-C Base Pairing 1) How many hydrogen bonds?
1) 3.
Nucleic Acids (Definition) 1) What is a nucleic acid? 2) Components of nucleic acids? 3) RNA is called? 4) DNA is called?
1) A biopolymer containing three types of monomer units. 2) A base derived from purine or pyrimidine (nucleases). A monosaccharide (either D-ribose or 2-deoxy-D-ribose). Phosphoric acid. 3) RNA (Ribonucleic Acid). 4) DNA (Deoxyribonucleic Acid)
DNA: 1° Structure 1) What is DNA? 2) What is the primary structure?
1) A biopolymer that consists of a backbone of alternating units of 2-deoxy-D-ribose and phosphate. The 3'-OH of one 2-deoxy-D-ribose is joined to the 5'-OH of the next 2-deoxy-D-ribose by a phosphodiester bond. 2) The sequence of bases along the pentose phosphodiester backbone of a DNA molecule base sequence is read from the 5' end to the 3' end.
Nucleosides 1) What is a nucleoside? 2) What do they lack? 3) Right carbon on the sugar is called?
1) A compound that consists of D-ribose or 2-deoxy-D-ribose covalently bonded to a nucleobase by a beta-N-glycosidic bond. 2) Lack phosphate groups. 3) Anomeric Carbon.
Nucleotides 1) What is a nucleotide? 2) Polymerization of nucleotides lead to what?
1) A nucleoside in which a molecule of phosphoric acid is esterified with an -OH of the monosaccharide. Most commonly either the 3'-OH or the 5'-OH. 2) Lead to nucleic acids. Linkage is repeated (3',5'-phosphodiester bond).
Chromatin 1) Each bead is? 2) Nucleosome consists of?
1) A nucleosome. 2) DNA wrapped around histone core.
Covalent Structure of Polynucleotides
1) Addition of phosphate groups to nucleotides gives rise to nucleotides, which are the monomers of nucleic acids. 2) When nucleotides are joined by phosphodiester bonds, they form a sugar-phosphate backbone, giving rise to DNA and RNA. 3) The sequence of bases is a very important feature of the primary structure of nucleic acids, because the sequence is the genetic information that ultimately leads to the sequence of RNA and protein.
Purine Bases 1) Consist of? 2) They are good what? 3) Adenine pKa at N1 is? 4) Guanine pKa at N7 is?
1) Adenine and Guanine. 2) Also good H-bond donors and acceptors. 3) 3.8. 4) 2.4.
Other Features of DNA 1) Bases are what? So how do they interact? 2) Bases in the minor groove in B-DNA are in contact with what? 3) A propeller-twist conformation leads to what? 4) B-DNA base-pair around what? Where does this occur?
1) Bases are hydrophobic and interact by hydrophobic interactions. 2) Come in contact with water 3) Base pairing distances are less optimal but base stacking is more optimal and water is eliminated from minor groove contacts. 4) B DNA base pair rotation around the axis of rotation. Occurs naturally, not in solution.
Comparison of A,B, and Z forms of DNA 1) Which ones form right-handed helices? 2) Which one forms left-handed helices? 3) Which one occurs in nature? What is also found in this?
1) Both A and B-DNA. 2) Z-DNA. 3) Z-DNA occurs in nature, usually consists of alternating purine-pyrimidine bases. Methylated cytosine found also in Z-DNA.
Super DNA Coiled Topology 1) Prokaryotic DNA is? What can it form?
1) Circular. It can form supercoils..
T-A Base Pairing 1) Base pairing is? 2) What plays a role in double helix stabilization? 3) T-A forms how many hydrogen bonds?
1) Complimentary. 2) Base pairing by hydrogen bonding between T-A and between C-G. 3) 2 hydrogen bonds.
Other Forms of DNA 1) B-DNA is considered what? The helix turns which way? What is its diameter? How many base pairs per turn? 2) A-DNA turns which way? How many base pairs per turn? How is it different than B-DNA? 3) Z-DNA turns which way? Plays a role in what?
1) Considered the physiological form. It is a right-handed helix. It has a diameter 11Å. 10 base pairs per turn (34Å) of the helix. 2) A right-handed helix. 11 base pairs per turn of the helix. Thicker than B-DNA. Has not been found in vivo. 3) A left-handed double helix. Plays a role in gene expression.
Kinds of RNA and Their Structures
1) Four kinds of RNA—transfer RNA, ribosomal RNA, messenger RNA, and small nuclear RNA—are involved in protein synthesis. 2) Transfer RNA transports amino acids to the sites of protein synthesis on ribosomes, which consist of ribosomal RNAs and proteins. 3) Messenger RNA directs the amino acid sequence of proteins. Small nuclear RNA is used to help process eukaryotic mRNA to its final form. 4) RNA interference, which requires short stretches of siRNA, exerts control over gene expression.
Principal Kinds of RNA 1) RNA consists of? 2) The pentose unit is? 3) Strands of RNA are?
1) Long, unbranched chains of nucleotides joined by phosphodiester bonds between the 3'-OH of one pentose and the 5'-OH of the next. 2) Beta-D-ribose (it is 2-deoxy-D-ribose in DNA). 3) RNA is single stranded (DNA is double stranded).
Z-Form is a Derivative of B-Form 1) Z-DNA is formed how? Without disturbing what?
1) Produced by flipping one side of the backbone 180˚ without disturbing the backbone covalent bonds or hydrogen bonds.
Information Transfer in Cells 1) Information encoded in the nucleotide sequence of DNA is transcribed through?
1) RNA synthesis.
DNA differs from RNA 1) The sugar in DNA is? As opposed to?
1) Sugar is 2'-deoxyribose, not ribose.
Denaturation of DNA 1) Denaturation is the disruption of what? 2) Denaturation is commonly done by? 3) As strands separate, absorbance increases where? What is this called? 4) Midpoint of transition (melting) curve is? 5) The higher the % G-C, the higher the? 6) When is renaturation possible? 7) Tm depends on?
1) The 2° structure. 2) Heat denaturation (melting). 3) Absorbance at 260 nm increases. Increase is called hyperchromicity. 4) Tm. 5) Tm 6) On slow cooling and annealing. 7) pH, Ionic Strength, Size and Base Composition.
Supercoiling in Eukaryotic DNA 1) Histones are rich in? Found in which organisms? 2) The 5 types of histones are? 3) What is the chromatin?
1) The basic amino acids Lys and Arg. Found associated with eukaryotic DNA 2) H1, H2A, H2B, H3, H4. Come together to form octamers. 3) DNA molecules wound around particles of histones in a beadlike structure. Topological changes induced by supercoiling accommodated by histone-protein component of chromatin.
Structure of DNA
1) The double helix is the predominant secondary structure of DNA. The sugar-phosphate backbones, which run in antiparallel directions on the two strands, lie on the outside of the helix. Pairs of bases, one on each strand, are held in alignment by hydrogen bonds. 2) The base pairs lie in a plane perpendicular to the helix axis in the most usual form of the double helix, but there are variations in structure. 3) The tertiary structure of DNA depends on supercoiling. In prokaryotes, the circular DNA is twisted before the circle is sealed, giving rise to supercoiling. In eukaryotes, the supercoiled DNA is complexed with proteins known as histones.
Nucleic Acids 1) Primary structures consist of? 2) The secondary structures consist of? 3) The tertiary structure consists of? 4) The quaternary structures consist of?
1) The order of bases on the polynucleotide sequence. The order of bases specifies the genetic code. 2) The three-dimensional conformation of the polynucleotide backbone. 3) Supercoiling. 4) Interaction between DNA and proteins.
Summary of Nucleic Acids 1) Primary structure refers to? 2) Secondary structure refers to? 3) Tertiary structure is?
1) The order of bases. 2) Three-dimensional conformation of the backbone. 3) The tertiary structure is the supercoiling of the molecule.
DNA: 2° Structure 1) What is the secondary structure? 2) What is an example of a 2° Structure? 3) What is the double helix? Two strands twist which way? Coiled which way?
1) The ordered arrangement of nucleic acid strands. 2) The double helix. 3) A type of 2° structure of DNA molecules in which two antiparallel polynucleotide strands are coiled in a right-handed manner about the same axis.
DNA: 3° Structure 1) What is the tertiary structure? 2) What is circular DNA? 3) Seen in which organisms? 4) What is supercoiling? 5) Supercoiling Topoisomerase Class 1 does? 6) Supercoiling Topoisomerase Class 2 does? 7) DNA Gyrase is?
1) The three-dimensional arrangement of all atoms of a nucleic acid. Commonly referred to as supercoiling. 2) A type of double-stranded DNA in which the 5' and 3' ends of each stand are joined by phosphodiester bonds 3) Seen in prokaryotes. 4) Further coiling and twisting of DNA helix. 5) Cut the phosphodiester backbone of one strand, pass the end through, and reseal. 6) Cut both strands, pass some of the remaining DNA helix between the cut strands, and reseal. 7) A bacterial topoisomerase.
Denaturation of DNA
1) The two strands of the double helix can be separated by heating DNA samples. This process is called denaturation. 2) DNA denaturation can be monitored by observing the rise in ultraviolet absorption that accompanies the process. 3) The temperature at which DNA becomes denatured by heat depends on its base composition; higher temperatures are needed to denature DNA rich in G-C base pairs.
Pyrimidine Bases 1) Base that's only found in RNA is? 2) Cytosine, Uracil and Thymine are all good what? 3) Cytosine pKa at N3 is? 4) Thymine pKa at N3 is?
1) Uracil is found only in RNA. 2) All are good H-bond donors and acceptors. 3) 4.5. 4) 9.5.
B-DNA in nature Propeller Twists 1) Bases in the minor groove come in contact with what? 2) As such, how do they twist? 3) Propeller twist results in what?
1) Water. 2) Propeller twist. 3) Less optimal base pair distance. More optimal base pair stacking (eliminates presence of water molecules).
10. Reflect and Apply In the early days of molecular biology, some re- searchers speculated that RNA, but not DNA, might have a branched rather than linear covalent structure. Why might this speculation have come about? 11. Reflect and Apply Why is RNA more vulnerable to alkaline hydrolysis than DNA?
10. This speculation arose from the fact that ribose has three hy- droxyl groups that can be esterified to phosphoric acid (at the 2', 3', and 5' positions), whereas deoxyribose has free hydroxyls at the 3' and 5' positions alone. 11. The hydrolysis of RNA is greatly enhanced by the formation of a cyclic 2',3'-phosphodiester intermediate. DNA, lacking the 2'-hydroxyl group, cannot form the intermediate and thus is relatively resistant to hydrolysis.
18. Recall How do the major and minor grooves in B-DNA compare to those in A-DNA? 19. Recall Which of the following statements is (are) true? (a) The two strands of DNA run parallel from their 5' to their 3' ends. (b) An adenine-thymine base pair contains three hydrogen bonds. (c) Positively charged counterions are associated with DNA. (d) DNA base pairs are always perpendicular to the helix axis.
18. The major groove and minor groove in B-DNA have very dif- ferent dimensions (width); those in A-DNA are much closer in width. 19. Statement (c) is true. Statements (a) and (b) are false. Statement (d) is true for the B form of DNA but not for the A and Z forms.
42. Recall What is the purpose of small nuclear RNA? What is an snRNP? 43. Recall Which type of RNA is the biggest? Which is the smallest? 44. Recall Which type of RNA has the least amount of secondary structure?
42. Small nuclear RNA (snRNA) is found in the eukaryotic nucle- us and is involved in splicing reactions of other RNA types. An snRNP is a small nuclear ribonucleoprotein particle. A complex of small nuclear RNA and protein catalyzes splicing of RNA. 43. Ribosomal RNA (rRNA) is the largest. Transfer RNA (tRNA) is the smallest. 44. Messenger RNA (mRNA) has the least amount of secondary structure (hydrogen bonding).
51. Reflect and Apply Explain briefly what happens to eukaryotic mRNA before it can be translated to protein. 52. Reflect and Apply Explain why a 50S ribosomal subunit and a 30S ribosomal subunit combine to form a 70S subunit, instead of an 80S subunit.
51. Eukaryotic mRNA is initially formed in the nucleus by transcrip- tion of DNA. The mRNA transcript is then spliced to remove introns, a poly-A tail is added at the 3' end, and a 5'-cap is put on. This is the final mRNA, which is then transported, in most cases, out of the nucleus for translation by the ribosomes. 52. The numbers 50S, 30S, etc. refer to a relative rate of sedimenta- tion in an ultracentrifuge and cannot be added directly. Many things besides molecular weight influence the sedimentation characteristics, such as shape and density.
9. Reflect and Apply A friend tells you that only four different kinds of bases are found in RNA. What would you say in reply?
9. Four different kinds of bases—adenine, cytosine, guanine, and uracil—make up the preponderant majority of the bases found in RNA, but they are not the only ones. Modified bases occur to some extent, principally in tRNA.
40. Reflect and Apply Why does DNA with a high A!T content have a lower transition temperature, Tm, than DNA with a high G!C content?
A-T base pairs have two hydrogen bonds, whereas G-C base pairs have three. It takes more energy and higher temperature to disrupt the structure of DNA rich in G-C base pairs.
22. Recall What is an AG/CT step?
An AG/CT step is a small section of double-stranded DNA where one strand is 5'-AG-3', and the other is 5'-CT-3'. The exact na- ture of such steps greatly influences the overall shape of a dou- ble helix.
30. Reflect and Apply Explain, and draw a diagram to show, how acety- lation or phosphorylation could change the binding affinity be- tween DNA and histones.
Histones are very basic proteins with many arginine and lysine residues. These residues have positively charged side chains under physiological pH. This is a source of attraction between the DNA and histones because the DNA has negatively charged phosphates: Histone-NH+ attracts ⁻O-P-O-DNA chain. When the histones become acetylated, they lose their posi- tive charge: Histone!NH!COCH3. They therefore have no at- traction to the phosphates on the DNA. The situation is even less favorable if they are phosphorylated because now both the histone and the DNA carry negative charges.
28. Reflect and Apply List three mechanisms that relax the twisting stress in helical DNA molecules. 29. Reflect and Apply Explain how DNA gyrase works.
Negative supercoiling, nucleosome winding, Z-form DNA. 29. It binds to the DNA, forming loops around itself. It then cuts both strands of DNA on one part of the loop, passes the ends across another loop, and reseals.
25. Recall If circular B-DNA is positively supercoiled, will these super- coils be left- or right-handed?
Positive supercoils in circular DNA will be left-handed.
21. Recall What is propeller-twist?
Propeller-twist is a movement of the two bases in a base pair away from being in the same plane.
23. Recall Why does propeller-twist occur?
Propeller-twist reduces the strength of the hydrogen bond but moves the hydrophobic region of the base out of the aqueous environment, thus being more entropically favorable.
20. Recall Define supercoiling, positive supercoil, topoisomerase, and negative supercoil
Supercoiling refers to twists in DNA over and above those of the double helix. Positive supercoiling refers to an extra twist in DNA caused by overwinding of the helix before sealing the ends to produce circular DNA. A topoisomerase is an enzyme that induces a single-strand break in supercoiled DNA, relaxes the supercoiling, and reseals the break. Negative supercoiling refers to unwinding of the double helix before sealing the ends to produce circular DNA.
35. Reflect and Apply What would be the most obvious characteristic of the base distribution of a single-stranded DNA molecule?
The base distribution would not have A=T and G=C, and total purine would not be equal to total pyrimidine.
45. Recall Why does the absorbance increase when a DNA sample unwinds?
The bases in a double-stranded chain are partially hidden from the light beam of a spectrophotometer by the other bases in close proximity, as though they were in the shadow of the other bases. When the strands unwind, these bases be- come exposed to the light and absorb it; therefore, the absor- bance increases.
33. Reflect and Apply What is the complete base composition of a dou- ble-stranded eukaryotic DNA that contains 22% guanine?
The percentage of cytosine equals that of guanine, 22%. This DNA thus has a 44% G-C content, implying a 56% A-T content. The percentage of adenine equals that of thymine, so adenine and thymine are 28% each.
Give the sequence on the opposite strand for ACGTAT, AGATCT, and ATGGTA (all read 5' l 3').
The sequence on the opposite strand for each of the follow- ing (all read 59 S 39) is ACGTAT TGCATA AGATCT TCTAGA ATGGTA TACCAT.