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BioChem > Chapters 4, 5, 8 COPY COPY > Flashcards

Chapters 4, 5, 8 COPY COPY Flashcards

1
Q

Name the two classes of nucleic acids and describe how they differ.

A

Deoxyribonucleic acid and Ribonucleic acid. Deoxyribonucleic acid has H on its 2’ carbon instead of OH. Deoxyribonucleic acid is more durable because it does not have the reactive OH. Ribonucleic acids -OH near the phosphate allows for the breakage of the phosphodiester bond

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2
Q

What are the four subunits of a nucleic acid?

A

Sugar, Heterocyclic Base, Phosphate, and Sugar-Phosphate backbone

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3
Q

Describe the sugar subunit of DNA, differentiate between the types

A

Five-membered ring, the carbons are described with a prime (‘), always has a 3’ OH group, the 2’ carbon has either OH or H

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4
Q

Describe the heterocyclic base subunit of DNA, differentiate between the types

A

Either Purine (Adenine or Guanine) or Pyrimidine. Purines have a hexagonal-pentagonal structure and are connected to the sugar by their N9 nitrogen Pyrimidines (Cytosine, Uracil, or Thymine) have a hexagonal structure and are connected to the sugar by their N1 nitrogen

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5
Q

What is the structure of Adenine?

A

-NH2 group connected to hexagonal ring of hexagon-pentagon

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6
Q

What is the structure of Guanine?

A

=O group and -NH2 groups connected to hexagonal ring of hexagon-pentagon

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7
Q

What is the structure of Cytosine?

A

One =O group, one -NH2 group. Single Hexagonal ring

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8
Q

What is the structure of Uracil?

A

Two =O groups only. Single Hexagonal ring

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9
Q

What is the structure of Thymine?

A

Two =O groups, one CH3 group. Single Hexagonal ring

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10
Q

What do we mean by N1 and N9 nitrogen? Which nitrogen is the N1 one and how are the rest of them numbered? (IMAGE)

A
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11
Q

What do we mean by N1 and N9 nitrogen? Which nitrogen is the N1 one and how are the rest of them numbered?

A

The order in which the carbons are labeled, N1 refers to the nitrogen at the first carbon, N9 the nitrogen at the last carbon (for purines) The N1 nitrogen is the NH opposite of either the =O or the -NH2 on the hexagonal ring for pyrimidines (next to the constant =O), OR the nitrogen furthest from the pentagonal ring for purines. The rest of the carbons are numbered so the second nitrogen is N3. For purines, the hexagonal group is numbered first, the connected carbons are 4 and 5 and the NH group on the pentagon is N9 (Counter-clockwise)

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12
Q

Describe the phosphate group subunit of DNA

A

Attached to the 5’ carbon Nucleoside is nucleotide without phosphate

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13
Q

Describe the phosphate-sugar backbone of DNA. What is this bond called and how does it form?

A

Phosphate attaches to 5’ carbon of one sugar and 3’ carbon of the other. Bases face inside the backbone. Phosphodiester bond, two strong covalent bonds form between the phosphate group and two sugars. The phosphate group is negatively charged and repels nucleophilic species, it is resistant to hydrolytic attacks.

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14
Q

What is the connection between the base and the sugar called?

A

b- glycosidic

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15
Q

What type of interactions occur between bases?

A

Hydrogen bonding, van der Waals, and hydrophobic interactions

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16
Q

How are DNA and RNA charged?

A

Negatively charged/acidic due to phosphate group

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17
Q

How do electrostatic interactions neutralize P?

A

Histones, cations, polyamines

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18
Q

What are some roles of nucleic acids?

A

Energy currency (ATP), metabolism, hormones, part of co-enzymes (where they carry electrons)

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19
Q

Where is there a UV absorption for DNA and RNA?

A

240-275 range due to conjugated bases

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20
Q

Describe the Watson and Crick structure of DNA

A

Adjacent bases separated by 3.4 AO, Helical structure repeats every 35.4 AO, 10.4 bases per turn, diameter of 20 AO, gap of major groove is 12 AO, gap of minor groove is 6 AO, glycosyl bond conformation is anti, right-handed Anti is most of the base inside the helix away from 5’

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21
Q

What are the two other forms of DNA?

A

A-form and Z-form

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22
Q

Describe the A-form of DNA

A

Dehydrated, right-handed, wider and shorter due to shape of ribose rings

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23
Q

Describe the Z-form of DNA

A

Left-handed with zigzagged phosphates, gene regulation

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24
Q

What is the suffix for nucleotides?

A

-ylate

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25
Q

What are the suffix for nucleosides?

A

-sine for AG bases, -ine for CUT bases

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26
Q

What is the prefix for DNA?

A

deoxy-

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27
Q

What does the Deoxyribonuclease enzyme do?

A

hydrolyzes phosphodiester bonds that link nucleotides

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28
Q

What does the Ribonuclease enzyme do?

A

Degrades RNA into smaller compounds

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29
Q

What does the Endonuclease enzyme do?

A

Type of deoxyribonuclease that cleaves in the middle of a DNA molecule

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30
Q

What does the Exonuclease enzyme do?

A

Type of deoxyribonuclease that cleanse at the end of a DNA molecule

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31
Q

What does the restriction endonuclease do?

A

Types of endonuclease that binds to a specific restriction site

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32
Q

What happened in the Avery, Macleod, and McCarty experiment? What does this mean?

A

Destroyed either Polysaccharides, Lipid, RNA, Protein, or DNA of dead S strain bacteria (deadly). Mixed each strain separately with live R strain bacteria (harmless). Only with the DNA destroyed did the mice survive contact with the bacteria mixture.

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33
Q

What happened in the Chargaff experiment?

A

Determined base composition in species -> Guanine # = Cytosine #, Adenine # = Thymine #. Relative amounts of each base vary from one species to another, which hinted that DNA could be the genetic material

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34
Q

What happened in the Hershey and Chase experiment?

A

Took bacteriophages and labeled DNA with phosphate and Protein with sulfur. After centrifugation with each, only phosphorus appeared in the cells.

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35
Q

What happened in the Watson and Crick experiment?

A

X-ray diffraction pattern of DNA from Rosalind -> described right handed, antiparallel double helix (B-DNA)

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36
Q

Describe the palindromic nature of DNA. How does this function

A

Segment of DNA on one strand is a palindrome with adjacent segment on the opposite strand (TTAGCAG vs GTGCTAA) Functions in Gene regulation. Creates hairpin/stem-loop or Cruciform (double hairpin)

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37
Q

Describe how DNA replication is semiconservative.

A

Half of the molecule is conserved in each generation

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38
Q

Who proved the semiconservative nature of DNA replication?

A

Meselson and Stahl using banding of 15N and 14N

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39
Q

How were DNA strands separated in the lab?

A

Disruption of the hydrogen bonds with heat and/or acid/alkali

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40
Q

Describe Tm. What changes it?

A

Melting Temperature, the temperature at which half of the helical structure of DNA is lost. Higher GC content = higher temperatures Tm depends on pH, ionic strength, and the size of the DNA

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41
Q

How can we detect whether DNA is double stranded or single stranded through UV Absorbance?

A

At 260 nm is the greatest difference between UV absorption between double-stranded and single-stranded DNA

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42
Q

What does Helicase do?

A

Uses ATP to disrupt the hydrogen bonds between base pairs

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43
Q

What do gyrase and topoisomerase do?

A

Relieves the tension created by the unraveling of the DNA double helix

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44
Q

What do single stranded binding proteins do?

A

Binds to single stranded regions of DNA to stabilize. Modulates the functions of numerous proteins involved in the processes of replication, recomibination, and repair.

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45
Q

Describe the chemistry of DNA Polymerase and what it requires to function

A

DNA is made in the 5’ to 3’ direction The 5* group of three phosphates from an incoming dNTP reacts with the 3*OH of the previous nucleotide, releasing two phosphates and attaching to the OH DNA Polymerase therefore requires an RNA primer with a free 3*OH already base-paired to the template

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46
Q

Describe the use of a primer in DNA synthesis. What enzymes are used in this process?

A

DNA Polymerase requires a free 3’OH, Primase is an RNA polymerase that synthesizes short stretches of RNA complementary to the template DNA strand that are later removed via hydrolysis and replaced with DNA by DNA polymerase. The gaps are sealed with ligase

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47
Q

What things are required for DNA replication?

A

DNA polymerase, DNA template, Primers (Primase), Ligase dNTPs and Mg+2

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48
Q

Describe DNA Polymerase

A

Has a “palm” active site and “fingers” and a ”thumb” to hold the DNA High catalytic power High fidelity (low error rate) Processive (Can catalyze many consecutive reactions without releasing its substrate)

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49
Q

Describe the DNA Template required for DNA replication

A

Both strands of DNA serve as templates, the site of dNA synthesis is the replication fork. DNA is then synthesized in a leading strand and a lagging strand

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50
Q

Describe a leading strand and lagging strand

A

A leading strand synthesizes DNA continuously in the direction of the replication fork, only requires one RNA primer. A lagging strand synthesizes DNA semidiscountinously and in the opposite direction of the replication fork in short “Okazaki fragments”, requires many RNA primers

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51
Q

What are topoisomers? What are linking numbers?

A

Circular DNA molecules with the same nucleotide sequence but different linking numbers Linking numbers are the number of times one strand of DNA is linked with the other in a covalently closed circular molecule OR the number of times one strand of DNA would have to be passed through the other in order for the two strands to be separated from each other

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52
Q

What is Lk?

A

Linking number Number of base pairs/10.4 (bp/turn). Always an integer Lk = Tw (twisting number) + Wr (writhing number)

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53
Q

What is Tw?

A

Twisting number- number of times the strands are twisted about each other

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54
Q

What is Wr?

A

Writhing number- measures the coiling fo the axis of the double integer

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55
Q

What is true about the equation Lk = Tw + Wr for a relaxed DNA molecule?

A

Lk = Tw (no supercoils)

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56
Q

Define negative supercoils and positive supercoils

A

Negative supercoils have a lower linking number than relaxed DNA and positive supercoils have a higher linking number than relaxed DNA

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57
Q

How does supercoiling affect DNA?

A

Compacts DNA for easy packaging and changes its interactions with other molecules

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58
Q

Describe the process of reverse transcription

A

RNA->DNA Synthesis of ssDNA complementary to one strand of RNA → Hydrolysis of ssRNA in the RNA/DNA hybrid → synthesis of the second strand of DNA Reverse transcriptases catalyze each of the three reactions

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59
Q

How is reverse transcriptase similar to DNA polymerase? How is it different?

A

Contain Zn+2, like DNA polymerase Lacks the 3*→5* proofreading

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60
Q

How do viruses infect hosts?

A

The genes of some viruses are made of RNA, and must be converted to DNA for insertion into the host chromosome Integrated viral genome is later expressed to form viral RNA and viral proteins

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61
Q

15% of genetic diseases are caused by what?

A

Mutations in RNA splicing

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62
Q

Define mRNA

A

Messenger RNA that serves as a template for protein synthesis

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63
Q

Define tRNA. What is its sedimentation coefficient(s)?

A

Transfer RNA carries amino acids to the ribosome At least one tRNA for each amino acid Sedimentation coefficient of 4

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64
Q

Define rRNA. What is its sedimentation coefficient(s)? What is its abundance in the cell?

A

Ribosomal RNA is the main component of ribosomes Sedimentation coefficients of 23, 16, and 5 Most abundant form of RNA at 80% (tRNA is 15% and mRNA is 5%)

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65
Q

Define snRNA

A

Small nuclear RNA that splices out RNA exons

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66
Q

Define Small RNA

A

Part of the signal-recognition particle, a RNA/Protein complex that guides new proteins

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67
Q

Define miRNA

A

Micro RNA, lab-made Small noncoding RNA that binds to complementary mRNA and inhibits translation

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68
Q

Define siRNA

A

Small interfering RNA that binds to mRNA and facilitates degradation

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69
Q

Where is RNA also found?

A

Telomerase

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70
Q

Which direction does DNA transcription occur?

A

5’ to 3’

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71
Q

Where does the transcription reaction occur?

A

Inside a 17bp (1.6 turn) span of unwound DNA known as the transcription bubble

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72
Q

Describe the two strands of DNA during transcription

A

DNA is said to have a template/antisense strand that is complementary to the new RNA and a coding/sense strand that has the same sequence

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73
Q

What are the requirements for transcription?

A

Requirements are a dsDNA template, activated precursors (NTPs), and a divalent metal ion (Mg+2 or Mn+2)

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74
Q

Define the three steps of RNA Transcription

A

Initiation- Unwinding of the DNA Elongation- Addition of nucleotides to the mRNA strand Termination- Halting of Elongation, the complete mRNA strand detaches from DNA

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75
Q

What does RNA polymerase do?

A

Initiation- binds to the promoter and signals the DNA to unwind Elongation- moves along the template DNA strand and synthesizes a complementary RNA strand Termination- Encounters a termination sequence, releases the RNA, and dissociates from the DNA

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76
Q

What is the sigma factor?

A

Subunit of bacterial RNA polymerase that is responsible for binding to the RNA Polymerase and enables binding of RNA Polymerase to the promoter

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77
Q

Describe RNA polymerase core enzyme vs holoenzyme

A

Core enzyme is the subunit of the enzyme needed for catalytic activity, 𝞪2𝜷𝜷‘ω When the sigma factor attaches it is known as a Holoenzyme, 𝞪2𝜷𝜷‘ωσ

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78
Q

What is the promoter?

A

Regions of DNA that bind RNA Polymerase and determine where transcription begins

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79
Q

What does the prokaryotic promoter look like? What does the eukaryotic?

A

Prokaryotes → Pribnow Box and -35 Region (-10,-35) Eukaryotes → TATA Box and CAAT Box (optional) (-25,-75)

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80
Q

How are the nucleotides of the mRNA sequence numbered?

A

The first nucleotide of the new mRNA is referred to as +1, the second one is +2. The the upstream nucleotide is called -1

81
Q

What is the mechanism of RNA polymerase?

A

Similar mechanism as DNA synthesis, incoming 𝞪 phosphate of NTP attaches to 3*OH of RNA chain

82
Q

Most new mRNA chains have what at their 5’ end?

A

pppG or pppA (triphosphate Guanine or Adenine)

83
Q

How is the RNA polymerase reaction thermodynamically favorable?

A

The creation of a phosphodiester bond releases a pyrophosphate (PP2) from the nucleotide triphosphate, which degrades into an orthophosphate (P + P)

84
Q

How is RNA Polymerase different from DNA polymerase?

A

Does not require a primer and does not proofread

85
Q

What type of structure does the newly synthesized RNA form?

A

Partial hybrid helix with the template DNA strand

86
Q

Which direction does RNA Elongation occur?

A

5’ to 3’

87
Q

Describe Rho-dependent termination

A

Controlled by the action of the Rho protein, an ATP-dependent (RNA/DNA) helicase that binds the RNA chain and pulls it from RNA P and the DNA template

88
Q

Describe Rho-independent termination

A

Regions of DNA contain stop signals that are transcribed into RNA as a palindromic GC rich region followed by an AT rich region Pairing between G/C forms a hairpin structure followed by many U’s RNA Polymerase pauses after the hairpin, allowing the weakly bound RNA to dissociate from the DNA and RNA Polymerase

89
Q

Describe RNA modification. Is mRNA modified?

A

mRNA is not modified, it may be translated as it is being transcribed.

90
Q

How are tRNA and rRNA created?

A

Cleavage from a single RNA

91
Q

What occurs to the termini of some RNA through modification? Specifically for tRNA?

A

Some termini get additional RNA, CCA is added to the 3’ ends of all tRNA

92
Q

Describe RNA Methylation

A

Modification of RNA to allow for protection from enzymes

93
Q

Describe why bases may be converted in RNA modification

A

Some of the bases in RNA can be converted to other bases, allows for structural/functional versatility Ex: tRNA has converted bases such as Inosole, which is ok because of wobble pairing

94
Q

Describe the overall process of RNA translation

A

Translating the sequence of a mRNA molecule to a sequence of amino acids during protein synthesis

95
Q

Describe Codon Degeneracy

A

Degeneracy of codons is the redundancy of the genetic code (multiple three-base pair codon combinations for a specific amino acid)

96
Q

How many codons code for Trp? Met? Stop?

A

Only one for Trp. Only one for Met (AUG). Stop codon has three (UAA/UGA/UAG)

97
Q

What determines the amount of codons for each amino acid?

A

The frequency of the amino acid in proteins

98
Q

Describe the prevalence of the standard amino acid code. How does it differ by organism?

A

The amino acid code is near-universal. Mitochondria and 16 other organisms have different codes

99
Q

How is the codon recognized?

A

Codon is complementary with anticodon of a specific tRNA, base pairs with H-bonds

100
Q

Describe Wobble base pairing.

A

RNA/DNA is always read 5* to 3*, so the third base on mRNA corresponds to the first base on the tRNA → This base is less important in binding

101
Q

Which three anticodon first bases can code for multiple bases?

A

U, G, or I (Inosine)

102
Q

How does wobble affect the speed of protein synthesis?

A

Allows for faster protein synthesis due to dissociation of tRNA

103
Q

Describe the tRNA and its 5’ and 3’ ends.

A

Clover-shaped, has an amino acid attachment site on its 3’ arm where its CCA sequence can bind with the carbonyl of the amino acid. The 5’ end is phosphorylated with pG (usually around 20, sometimes less)

104
Q

How does the amino acid bind to tRNA, which enzyme is involved?

A

Individual aminoacyl-tRNA synthetases attach each amino acid to the 3’ side of tRNA using ATP

105
Q

What is AA to tRNA known as?

A

The “second genetic code”

106
Q

What are the groups of unpaired bases that tRNA contains?

A

Clockwise when the CCA is on the top right: its terminal CCA the TψC loop the extra arm the anticodon loop the D arm

107
Q

Describe the extra arm of tRNA

A

Between the the TψC loop and the anticodon loop, variable in size and not always present

108
Q

Which direction is mRNA read and proteins synthesized? What does this allow for?

A

mRNA is read 5’ to 3’ and protein is made amino to carboxyl. This allows for proteins to be made while mRNA is being transcribed in some prokaryotes

109
Q

What occurs on the ribosome for initiation?

A

mRNA and aminoacylated tRNA bind to the ribosome The small subunit has a 16S section that recognizes the start codon The Shine-Dalgarno sequence in mRNA pairs with rRNA ins 16s subunit Large subunit has a 23s that forms the peptide bond

110
Q

What are the ribosomal subunits

A

70S ribosome is made of a small (30S) and large (50S) subunit

111
Q

Initiation of protein transcription is aided by how many initiation factors?

A

3

112
Q

Give an overview of protein synthesis.

A

The small ribosomal subunit recognizes the start codon and the large ribosomal subunit synthesizes the peptide bonds. Once mRNA binds to the small subunit, tRNA finds the P site and attaches fMET. The large subunit then joins with the small subunit. A new tRNA-AA comes in and attaches to the A site, the large subunit forms a peptide bond. The ribosome moves down the mRNA, powered by GTP. The P site tRNA detaches from the amino acid and moves to the exit site, where it leaves

113
Q

Give an overview of the initiation of protein synthesis.

A

The small ribosomal subunit recognizes the start codon (through the upstream Shine-Dalgarno sequence) and the large ribosomal subunit synthesizes the peptide bonds. Once mRNA binds to the small subunit, tRNA finds the P site and attaches fMET. The large subunit then joins with the small subunit.

114
Q

Give an overview of the elongation of protein synthesis (At this point there is a tRNA-fMET at the P site and the two ribosomal subunits are combined)

A

A new tRNA-AA comes in and attaches to the A site, the large subunit forms a peptide bond. The ribosome moves down the mRNA, powered by GTP. As this occurs the P site tRNA detaches from the amino acid and moves to the E site, where it leaves. Elongation continues through cycles of aminoacyl-tRNA binding and peptide bond formation

115
Q

How many base pairs a second is DNA Synthesis?

A

800-1000

116
Q

How many base pairs a second is Transcription?

A

50

117
Q

What is the error rate of DNA synthesis?

A

1/10^ -8 to -10

118
Q

What is the error rate of Transcription?

A

1/10^ -4

119
Q

How many base pairs a second is Translation?

A

50

120
Q

What is the error rate of Translation?

A

1/10^ -4

121
Q

Give an overview of the termination of protein synthesis. How many releasing factors?

A

Stop codon (UAA/UGA/UAG) is reached, mRNA and protein dissociate and ribosome is recycled 3 releasing factors

122
Q

What occurs after termination of protein synthesis?

A

Folding and processing

123
Q

Which way did we discuss the function of antibiotics besides targeting protein synthesis?

A

Blocking the function of bacterial gyrases (novobiocin)

124
Q

Which ways do antibiotics target protein synthesis?

A

Puromycin has a similar structure to the 3* end of charged tRNA, blocks the A site and causes chain termination Tetracyclines block the A site on ribosomes Chloramphenicol and Cycloheximide block peptidyl transferase Chloramphenicol also inhibits mitochondrial and chloroplast ribosomes Streptomycin causes misreading of code and can inhibit initiation

125
Q

What do restriction endonuclease’s do?

A

Recognize specific sequences of DNA and cleave both strands

126
Q

What do the sequences of DNA that restriction endonucleases recognize look like?

A

Four to eight base pairs with two fold rotational symmetry (5’ - 3’ same on both strands)

127
Q

How are restriction enzymes named?

A

From their bacteria, genus, species, strain, and type

128
Q

What type of cuts can restriction enzymes make? Differentiate between them

A

Either staggered or straight cuts. Staggered cuts produce sticky ends that can be used to create rDNA Two pieces of DNA cut with the same restriction enzyme can be combined and ligated together Straight cuts make blunt ends that cannot be recombined

129
Q

Describe DNA Mapping

A

Repeated treatment of DNA with different restriction enzymes and separation by electrophoresis with ethidium bromide yields very small fragments that can be sequenced manually

130
Q

Describe Southern Blotting

A

Separated DNA from agarose gel is transferred with an alkaline solution onto nitrocellulose sheets. It is then labeled with a complementary 32P DNA probe. A rinse removes unbound probe. It can then be observed via autoradiography. The labeled area can now be found.

131
Q

Describe Northern Blotting

A

Same as Southern blotting but for RNA. Transfer occurs with salt.

132
Q

Describe Western Blotting

A

Protein. Probe is not complementary DNA but an antibody attached to a radioactive label or enzyme. Visualization can occur with autoradiography if a radioactive label was used as a marker. If an enzyme was used visualization can occur through a color change

133
Q

Define Sanger Sequencing or Chain Termination Sequencing

A

A way to sequence small sections of DNA using preemptive chain termination caused by the introduction of ddNTP

134
Q

What is required for Sanger Sequencing?

A

2’,3’ -Dideoxy analogs of each nucleotide (Nucleotides where the sugar has only a hydrogen at both the 2’ and 3’ positions)

135
Q

What information is needed to be known in order to sequence the target DNA in Sanger Sequencing

A

Small segments of DNA to be sequenced must be to the right (downstream) of known sequences so the proper primer can be developed

136
Q

Describe the traditional method of Sanger sequencing. What is the collection of fragments called?

A

Four tubes contain the DNA to be sequenced, dye-labeled primers, all four dNTPs, DNA Polymerase, and a specific concentration of one of the four ddNTPs Correct ratio of ddNTP and dNTPs ensures that chain termination only rarely occurs, so that many fragments of different sizes are created Dye or radioactive tag can also be on the ddNTPs Collection of fragments is called a sequencing ladder and is separated by four-lane electrophoresis

137
Q

How is the sequencing read in the traditional Sanger method?

A

Read bottom (5’) to top (3’) to read the complementary strand of the sequence strand. (The smaller fragments are towards the bottom)

138
Q

Describe the computer-aided method of Sanger sequencing

A

-A different fluorescent tag is added to the ddNTP for each tube -After fragments are produced, tubes are combined and contents are separated by capillary electrophoresis -Laser-assisted fluorescence detector reads color and sends information to a computer -Graph of fluorescence intensity and oligonucleotide length is produced for each of the four colors

139
Q

How is the sequencing read in the computer-aided Sanger method?

A

Left to right by the peaks. 5’ -3’ complementary strand.

140
Q

Define Polymerase Chain Reaction

A

Amplifies (copies) a region of DNA that lies between two regions of known sequences

141
Q

What is required for Polymerase Chain Reaction?

A

-Two oligonucleotide DNA primers complementary to the outer regions of the known sequence (do not have to match perfectly) -The DNA fragment to be denatured in order to serve as the template -dNTP and DNA polymerase to create the new DNA

142
Q

How many new strands does Polymerase Chain Reaction create?

A

2^n strands per cycle.

143
Q

Describe how the new strands form in Polymerase Chain Reaction?

A

The 5’ end of the new strand matches the 5’ oligonucleotide primer, which attached to the 3’ primer of the target strand. The 3’ end varies in length and there will be a mix of long and sequence length DNA.

144
Q

Describe the cycle of Polymerase Chain Reaction. How many cycles is typical?

A
  1. Strand Separation at 95* C 2. Hybridization of primers at 37*C to 54*C 3. DNA synthesis at 72*C (72*C is the optimum temperature of Taq DNA polymerase, derived from Thermus aquatics bacteria in hot springs) Usually around 30 cycles
145
Q

How many long and sequence length new strands will be produced during Polymerase Chain Reaction?

A

Each cycle produces 2 long strands and 2^n -2 sequence-length strands

146
Q

How does Reverse Transcriptase PCR (RT-PCR) differ from regular PCR?

A

Used to amplify RNA sequences, the first step includes reverse transcriptase

147
Q

How does Quantitative PCR (Q-PCR) differ from regular PCR?

A

Use of probes or dye and continuous detection allows for quantification of the amount of starting material

148
Q

What are the overall qualities of PCR?

A

Rapid, sensitive, and robust, with many useful variations. Limited product and length (2kbp)

149
Q

What is a Holoenzyme?

A

proteinaceous enzyme and non-protein cofactor

150
Q

What is an Apoenzyme?

A

protein portion of holoenzyme

151
Q

What is a Zymogen or Proenzyme?

A

Catalytically inactive enzyme complex that can be easily and rapidly activated when needed

152
Q

What is a Cofactor?

A

a metal or coenzyme that is needed for the catalytic activity of many enzymes

153
Q

What is a Coenzyme and what are the types?

A

a small organic molecule, often derived from vitamins Prosthetic Groups = tightly-bound coenzyme Co-Substrate = loosely-lound coenzyme

154
Q

What is a biochemical catalyst?

A

Can accelerate a chemical reaction without itself undergoing any net change

155
Q

How much can enzymes accelerate reactions?

A

Up to six orders of magnitude (10^6)

156
Q

Describe the Enzyme Classification System (EC)

A

Each enzyme has a four number designation, for class, subclass, sub-subclass, and the specific enzyme

157
Q

What is the classification oxidoreductases? Which class is it?

A

Gaining/Losing electrons, dehydrogenases (1)

158
Q

What is the classification transferases? Which class is it?

A

Group Transfer (2)

159
Q

What is the classification hydrolyses? Which class is it?

A

Hydrolysis, transfer of functional groups to water (3)

160
Q

What is the classification lyases or synthases? Which class is it?

A

Addition or removal of groups to form double bonds (4)

161
Q

What is the classification Isomerases? Which class is it?

A

Transfer groups within molecules (5)

162
Q

What is the classification ligase or synthetase? Which class is it?

A

Ligation of two substrates at the expense of ATP hydrolysis (6)

163
Q

What does the free energy difference (deltaG) between reactants and products determine?

A

If the reaction is spontaneous. If deltaG is negative the reaction is spontaneous/exergonic.

164
Q

What is the general equation for deltaG?

A

GProducts- GReactants

165
Q

What is deltaG at equilibrium?

A

0

166
Q

How much does deltaG depend on the path of the transformation?

A

It doesn’t

167
Q

What is Keq?

A

ratio of products to reactants at equilibrium, =KF/KR

168
Q

What unit is deltaG?

A

kJ/mol

169
Q

In a sequence of consecutive reactions how can you calculate deltaG?

A

Addition of the deltaG’s for each reaction

170
Q

What is activation energy? What is it proportional to and how is it affected?

A

The energy required to initiate the conversion of reactants into products Proportional to the rate of reaction and is affected by enzymes

171
Q

How do enzymes accelerate reactions?

A

Facilitating formation of the transition state (X‡), or in other words lowering the activation energy.

172
Q

What is the transition state in terms of free energy and stability? Is it an intermediate?

A

Highest free energy, least stable form. Not an intermediate

173
Q

What is the general equation for activation energy?

A

deltaG between the transition state and substrate

174
Q

Define active site

A

Active site is the region on an enzyme that bonds the substrate and possesses catalytic residues that participate in reaction mechanism

175
Q

What is an enzyme-substrate complex?

A

Temporary molecule formed when a substrate causes a conformational change in the enzyme and enters the active site

176
Q

Describe how reaction rates change at a constant concentration of enzyme but with varying levels of substrate

A

At a constant concentration of enzyme, reaction rates increase with added substrate until maximum velocity is reached → indicates ES is formed and is the limiting variable

177
Q

What does substrate interaction with active site promote?

A

Substrate transition state

178
Q

Describe the structure and size of the active site

A

Active site is a 3D cleft formed by groups found on different parts of the AA sequence. It only takes up a small part of the enzyme

179
Q

What makes up the rest of enzyme besides the active site?

A

Most of the enzyme is scaffold, regulatory sites, sites for protein interactions, and substate channels

180
Q

Describe the unique microenvironment created by the active site.

A

Non-polar, usually excludes water, polar residues have special properties

181
Q

How are substrates bound to the active site?

A

Many weak interactions. Electrostatic, hydrogen bonds, van Der Waals (optimized due to complementary shape)

182
Q

What does the binding specificity of the active site depend on?

A

atoms in active site

183
Q

Describe the lock and key model of an active site

A

Active site of unbound enzyme is complementary to substrate

184
Q

Describe the dynamic induced fit model of an active site

A

Active site assumes complementary shape after substrate is bound. Substrate induces conformational change to bring together catalytic and binding groups

185
Q

How do enzymes lower activation energy? How is this maximized

A

Using binding energy- the free energy released upon multiple weak interactions between enzyme and substrate. Maximized with the right substrate and conversation into the transition state.

186
Q

Define enzyme activity and give units

A

amount of substrate converted to product by enzyme per time (μM/min)

187
Q

Define specific activity

A

ratio of enzyme activity to the quantity of protein

(μM/ min*mg)

188
Q

What is the enzyme international unit?

A

International Unit: quantity of enzyme needed to transform 1.0 μM of substrate to product per minute at 30*C and optimal pH

189
Q

In words, how does the initial velocity relate to the enzyme and substrate concentrations?

A

The initial velocity (d[P]/dt) is linear with enzyme concentration, increases with substrate concentration at low levels, and approaches a maximum at high levels

190
Q

What are the assumptions for the Michaelis-Menten equation?

A

The reaction is at steady state (d[ES]/dt =0)

The first step is fast and at equilibrium

No back reaction of [P] to [ES]

191
Q

For the generic enzyme reaction: E + S ES -> P + E

Where the formation of ES is k1, the reverse reaction to E + S is k2, and the reaction to P + E is k3

How do you derive the Michaelis-Menton equation?

A

Steady State

k1 [E][S] = (k2 + k3) [ES]

[ES] = [E][S] / (k2 + k3)/k1

(k2 + k3)/k1 is KM

[ET] = [E] + [ES]

[ES] = [ET][S] / (KM + [S])

Initial and Maximum velocity

With assumptions, VI = k3 [ES] and VMAX = k3 [ET]

VI = VMAX[S] / (KM + [S])

Graph of Reaction Velocity by Substrate Concentration

When [S] <<< KM, initial velocity is directly proportional to [S]

At ½ VMAX, KM = [S]

When [S] >>> KM, VI = VMAX

192
Q

How does Km relate to the relationship between the enzyme and its substrate?

A

higher Km, weaker the interaction between the enzyme and substrate

193
Q

[REVERSE]

-NH2 group connected to hexagonal ring of hexagon-pentagon

A

What is the structure of Adenine?

194
Q

[REVERSE]

=O group and -NH2 groups connected to hexagonal ring of hexagon-pentagon

A

What is the structure of Guanine?

195
Q

[REVERSE]

One =O group, one -NH2 group. Single Hexagonal ring

A

What is the structure of Cytosine?

196
Q

[REVERSE]

Two =O groups only. Single Hexagonal ring

A

What is the structure of Uracil?

197
Q

[REVERSE]

Two =O groups, one CH3 group. Single Hexagonal ring

A

What is the structure of Thymine?

198
Q

What do the 16S, 23S, and 5S portions of the ribosome do?

A

16S - Small ribosome, recognizes Shine-Dalgarno

23S - forms peptide bond

5S - structural

BioChem (6 decks)

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