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Biochem > Exam3 Ch 8 And 9 > Flashcards

Exam3 Ch 8 And 9 Flashcards

1
Q

Nucleotides role in metabolism:

A

-energy currency for metabolism
-essential chemical links in cell responses to hormones and other stimuli
-structural components of enzyme cofactors and metabolic intermediates
-constituents of nucleic acids = DNA, RNA

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2
Q

Functions of DNA

A

Storage of biological info
Transmission of that info to next gen

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3
Q

What are the classes of RNA and their purpose

A

RRNA- components of ribosomes
MRNA- intermediates in protein synthesis
TRNA- adapters, they translate info in MRNA into an AA sequence
NC RNA- many functions (non coding)

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4
Q

Three components of nucleotides

A

-nitrogenous base (pyramiding or purine)
-Pentose
- +1 phosphates

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5
Q

What is a nucleoside

A

Molecule without phosphate

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6
Q

Nucleotide bonds

A

-N B glycosyl bond- covalently joins 1’ carbon of the pentose to the base (at N1 of pyrimidines and N9 of purines), and is formed by removal of water

-the phosphate is esterfied to the 5’ carbon

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7
Q

Which purine / pyrmidine bases are in DNA vs RNA

A

Purine: A and G are both in DNA and RNA

Pyrimidine: C is in DNA and RNA
T is in DNA only
U is in RNA only

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8
Q

The 2 types of nucleotide pentoses

A

2’ deoxy-d-ribose in DNA
D ribose in RNA

-both are in B-furanose form (closed 5 member ring)

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9
Q

What are the 4 major deoxyribonucleotides (structural units of DNA)

A

-deoxyadenylate
-deoxyguanylate
-deoxythymidylate
-deoxycytidylate

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10
Q

What are the 4 major ribonucleotides (structural units of RNA)

A

-adenylate
-guanylate
-uridylate
-cytidylate

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11
Q

Examples of nucleotides with phosphate groups in different positions

A

-ribonucleoside 2’3’-cyclic monophosphate
-ribonucleoside 3’ monophosphate (made byRNA hydrolysis)
-cAMP (adenosine 3’5’ cyclic monophosphate)
-cGMP (guanosine 3’5’ cyclicmonophosphate)

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12
Q

Phosphodiester linkage

A

Covalent bond that joins successive nucleotides of both DNA and RNA

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13
Q

Hydrolysis of DNA and RNA (under alkaline conditions)

A

RNA is rapidly hydrolyzed due to 2’ hydroxyl groups
-DNA is not rapidly hydrolyzed \

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14
Q

What is oligonucleotide and polynucleotide

A

Oligo- short (<50 nt) nucleic acid
Poly- longer nucleic acid

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15
Q

Describe nucleotide bases

A

Weakly basic compounds.
-aromatic
-pyrimidines are planar, purines have pucker

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16
Q

Free pyrimidine and purine bases may exist as…

A

Tautomers
-lactam, lactim, double lactim

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17
Q

Nucleotides and UV absorption

A

All nucleotide bases absorb UV light, strong at 260nm

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18
Q

Describe solubility of nucleotides

A

Hydrophobic, insoluble in water, leads to VDW and dip dip interactions
-charged and more soluble at acidic or alkaline pH

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19
Q

Describe base pairs

A

H bonding patterns between complementary strands of nucleic acids , A to T or U, and G to C

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20
Q

Describe the levels of nucleic acid structure

A

Primary- covalent structure and nucleotide sequence
Secondary- regular, stable structure taken up by the nucleotides
Tertiary- complex folding of large chromosomes, folding of tRNA or rRNA structures

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21
Q

What did x ray diffraction reveal

A

DNA molecules are helical

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22
Q

Watson crick model of DNA

A

-major groove and minor groove
-3 H bonds between G and C
-2 H bonds between A and T

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23
Q

Phosphodiester bonds can… (in DNA strands )

A

Run in the same or opposite directions- parallel or antiparallel

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24
Q

Double helix has ____ bp per helical turn

A

10.5bp

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25
Q

Hydrogen bonding contributes to stability of DNA structure

A

Not really

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26
Q

Double helical DNA strands are…

A

Complementary

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27
Q

How is the DNA double helix stabilized

A

-metal cations that shield negative charges of backbone phosphates
-base stacking interactions between successive base pairs.
-duplexes with lots of GC are MORE stable

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28
Q

The 2 steps for replication of DNA

A
  1. Parent strands become separated
  2. Each parent strand serves as a template for synthesis of complementary daughter strand
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29
Q

Examples of structural variation in DNA

A

-different conformations of deoxyribose
-rotation about the phosphodeoxyribose backbone
-free rotation about the C-1’-N glycosyl bond

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30
Q

Describe the 3 forms of DNA

A

B form: (Watson crick)- most stable, random sequence
A form: right handed, wider, tilted plane, favored if no water
Z form: left handed, backbone with zig zag, more slender

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31
Q

Palindrome DNA occurs in

A

Regions with inverted repeats

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32
Q

What is a mirror repeat

A

Occurs when the inverted repeat is in each individual strand

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33
Q

Hairpin and cruciform structures occur from…

A

The self complementarity in each strand

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34
Q

DNA structures with 3 strands

A

Hoogsteen positions (N7, O6,, and N6 of purines)
Hoogsteen pairing- non- WC pairing
Triplex DNAs- form from Hoogsteen pairing

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35
Q

DNA structures with 4 strands

A

Tetraplex DNA -4 DNA strands pair, only when there’s a high level of G residues
G tetraplex= very stable

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36
Q

Transcription is..

A

Process of mRNAs forming on a DNA template.
MRNA- part of RNA carrying genetic info from DNA to ribosome

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37
Q

MRNAs code for

A

Polypeptide chains

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38
Q

MRNA can be. Monocistronic or polycistronic - describe

A

Mono- codes for one polypeptide (most mRNA in eukaryotes)
Poly- codes for 2+ polypeptides, in bacteria and archaea

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39
Q

MRNA is always ______ stranded (describe)

A

Single.
-with right handed conformation, and base stacking interactions
-can base pair w complementary DNA or RNA (paired strands are antiparallel)

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40
Q

Describe secondary structure of RNA

A

-complementary RNA strands form an A form right handed double helix
-breaks result in bulges or internal loops
-internal loops form palindromic sequences

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41
Q

Base paired helical structures in RNA

A

-base paired helical segments form in RNA
-hair pins most common

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42
Q

The off set pairing in 3D DNA structure creates…

A

Major and minor grooves on the surface

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43
Q

Desaturation of double helical RNA/DNA occurs by…

A

-high temp or extreme change in pH
-will disrupt H bonds and base stacking interactions

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44
Q

Double helical DNA/RNA can anneal (explain )

A

Annealing- 2 strands spontaneously rewind when Temp orPH Is returned to normal range

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45
Q

Hypochromic effect

A

Observed decrease in absorption of UV light when complementary strands are paired

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46
Q

Hyperchromic effect

A

Increase in absorption of UV light when a double stranded nucleic acid is denatured

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47
Q

Heat desaturation of DNA

A

Temp where 1/2 of DNA is separated single strands
-this increases with GC pairs

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48
Q

Partially denatured DNA is rich with ____ bp’s

A

AT

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49
Q

____ duplexes are more stable to heat denaturation than ___ duplexes

A

RNA, DNA

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50
Q

Nucleotides and nucleic acids undergo mutations which are…

A

Nonenzymatic transformations

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51
Q

Deamination

A

Spontaneous loss of exocyclic amino groups
-deamination of cytosine to uracil happens about 100 events/day, recognized as foreign in DNA and removed- reason why thymine occurs in DNA rather than uracil

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52
Q

Depurination

A

Hydrolysis of N-B-glycosyl bond between the base and Pentose , and creates an Apurinic site (AP)

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53
Q

Reactions made by radiation

A

UV light causes cyclobutane pyrimidine dimers
Ionizing radiation causes ring opening, base fragmentation, and breaks in covalent backbone of nucleic acids

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54
Q

DNA damage by reactive chemicals (give examples)

A

Nitrous acid precursors are deaminating agents
Alkylating agents = create modified nucleotides (non-enzymatically)

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55
Q

Alkylating agents can alter bases of DNA..

A

-they can methylate guanine to O6-methyl-guanine, which can’t base pair with cytosine

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56
Q

Explain DNA damage by oxidative damage

A

Reactive oxygen species (hydrogen peroxide, hydroxyl radicals, superoxide radicals) can damage DNA
-OH radicals are most common for oxidative DNA damage

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57
Q

Some bases of DNA are methylated…

A

A and C are methylated more than G and T
- all known DNA methylases use S-adenosylmethionine as a methyl group donor
- in. Eukaryotes, 5% of cytidine residues are methylated

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58
Q

Chemical synthesis of DNA by ________ method is highly efficient

A

Phosphoramidite

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59
Q

Describe PCR

A

-method of amplifying DNA segment of interest, relies on DNA polymerases (enzymes that make DNA from dNTPs using a DNA template)
-DNA polymerases add nucleotides to the 3’ ends of primers

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60
Q

Sanger sequencing

A

Dideoxy chain termination sequencing, and ddNTPs interrupt synthesis
-each four of the ddNTPs are labeled with a fluorescent tag

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61
Q

Describe reversible terminator sequencing

A

Uses 4 different modified deoxynucleotides (A,T,C,G), each with a certain fluorescent label and 3’ blocking group

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62
Q

Sequencing depth

A

Number of times a particular nucleotide in a genome is sequenced

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63
Q

Contigs

A

Long, contiguous sequences that are assembled from overlaps

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64
Q

Hydrolysis of ________ provide chemical energy (ATP)

A

nucleoside triphosphates

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65
Q

When coupled to a ________ ATP hydrolysis shifts eq to product

A

Positive free energy change

66
Q

Nucleotide binding fold

A

Single protein domain that binds adenosine

67
Q

Second messengers

A

Compounds made in the cell following interaction of extracellular chemical signals with receptors (cAMP)

68
Q

PpGpp

A

Made in bacteria during AA starvation to inhibit the synthesis of the rRNA and tRNA molecules

69
Q

Adenine nucleotides can serve as signals

A

ATP and ADP:
-neurotransmitters in a variety of signaling pathways
-signals for receptors that mediate pain sensation
-blood clotting signals

70
Q

Which nucleoside is commonly found as part of coenzymes

A

Adenosine

71
Q

How nucleotides be involved in signaling

A

Precursors for making second messengers
Extracellular ADP and ATP binding to its receptor

72
Q

Explain DNA Cloning

A

Selective amplification of a particular gene or DNA segment

73
Q

Explain recombinant DNA tech (genetic engineering)

A

Methods to do DNA cloning

74
Q

the 5 processes of DNA cloning

A

-obtain DNA segment to be cloned
-select molecule of DNA capable of autonomous replication
-join the 2 DNA fragments covalently
-move the recombinant DNA from the test tube to the host organism
-select the host cells that have the recombinant DNA

75
Q

what are cloning vectors and recombinant DNAs?

A

cloning vector- small DNA capable of autonomous replication
recombinant DNA- composite DNA made of covalently linked segments from 2 or more sources

76
Q

what are the advantages of cloning in E coli?

A

-DNA metabolism is well understood
-many naturally occuring cloning vectors like plasmids and bacteriophages
-techniques for moving DNA from one bacteria to another

77
Q

enzyme used in recombinant DNA tech:
Type 2 restriction endonuclease

A

cleaves DNA at specific base sequences

78
Q

enzyme used in recombinant DNA tech:
DNA ligase

A

joins 2 DNA molecules or fragments

79
Q

enzyme used in recombinant DNA tech:
DNA poly I

A

Fills gaps in duplexes by stepwise addition of nucleotides to 3’ ends

80
Q

enzyme used in recombinant DNA tech:
reverse transcriptase

A

makes a DNA copy of an RNA molecule

81
Q

enzyme used in recombinant DNA tech:
polynucleotide kinase

A

adds phosphate to the 5’ OH end of a polynucleotide to label it, or to help ligation

82
Q

enzyme used in recombinant DNA tech:
terminal transferase

A

adds homopolymer tails to the 3’ OH ends of a linear duplex

83
Q

enzyme used in recombinant DNA tech:
exonuclease III

A

removes nucleotide residues from the 3’ ends of a DNA strand

84
Q

enzyme used in recombinant DNA tech:
bacteriophage λ exonuclease

A

removes nucleotides from the 5’ ends of a duplex to expose single stranded 3’ ends

85
Q

enzyme used in recombinant DNA tech:
alkaline phosphotase

A

removes terminal phosphates from the 5’ end and 3’ end

86
Q

restriction endonuclease AKA restriction enzymes

A

recognize and cleave DNA at specific sequences (recognition sites)

87
Q

methylases

A

catalyze methylation of host DNA to protect it from digestion by the host cell’s restriction endonucleases

88
Q

restriction mod system

A

restriction endonuclease and the corresponding methylase

89
Q

types of restriction endonucleases

A

type 1 and 3: large multisubunit complexes with both endonuclease and methylases

type 2: simpler, requires no ATP, and catalyzes the hydrolytic cleavage of DNA phosphodiester bonds within the recognition sequence

90
Q

size of type 2 endonucleases

A

4-6 bp long
palindromic

91
Q

DNA ligase

A

joins DNA fragment to be cloned into a suitable cloning vector

92
Q

restriction endonucleases can leave either sticky ends or blunt ends (describe)

A

sticky ends- unpaired bases on the ends (due to staggered cuts, can base pair with comp sticky ends)

blunt ends- no unpaired bases on ends, straight cuts

93
Q

the DNA segment to be cloned is made by PCR, and including the _______ facilitates the cloning of amplified DNA

A

restriction endonuclease cleavage sites

94
Q

cleavage of PCR amplified DNA creates sticky ends used to…

A

ligate the amplified DNA to a cloning vector

95
Q

describe linkers and multiple cloning site

A

linker- synthetic DNA fragments created to bridge ligated ends
multiple cloning site- inserted DNA fragment that has multiple recognition sequences for restriction endonucleases

96
Q

what are the three cloning vectors

A

-plasmids
-bacterial artificial chromosomes
-yeast artificial chromosomes

97
Q

describe plasmids

A

circular DNA
-replicates seperately from the host chromosome
-5,000 to 400,000 bp
-symbiotic role in cell

98
Q

features of the E coli plasmid

A

-origin of replication: where replication is initiated
-resistance genes
-recognition sequences for restriction endonucleases

99
Q

describe bacterial transformation and electroporation

A

transformation- small plasmids are introduced into bacteria through heat shock (only successful w small plasmid)

electroporation- small plasmids are introduced to bacteria through high voltage pulses, transiently making the bacteria membrane permeable

100
Q

selectable markers and screenable markers are used to identify cells that take up plasmid DNA

A

selectable marker- permits growth of cell (positive selection) or kills the cell (negative selection)

screenable marker- gene encoding a protein that causes the cell to make a colored/flourescent molecule

101
Q

plasmid pBR322 makes __________ selection marker

A

positive and negative

102
Q

shuttle vectors

A

plasmids that can be propagated in cells of 2+ species , can incorporate multiple replication origins

103
Q

plasmid vectors allow for cloning of very long DNA

A

100,000 to 300,000 bp

104
Q

BACs are made up of…

A

made up of plasmid vector and large segments of cloned DNA

105
Q

features of the BAC vector

A
  • stable ORI’s
    -par genes from an F plasmid that direct the distribution of recombinant chromosomes at cell division
  • Cam R: positive selection marker
    -lac Z: screenable marker
106
Q

what is the YAC made up of…

A

plasmid vector and large segments of cloned DNA

DNA in YAC can be used to study: specialized sequence of chromosome metabolism
-mechanisms of gene regulation and expression

107
Q

the YAC vector has elements to maintain a eukaryotic chromosome in yeast nucleus:

A

-yeast ORI
-2 selectable markers
-specialized sequences for stability and chromosome segregation at cell division- the centromere and 2 telomeres

108
Q

stability of YAC clones…

A

increases with the length of the cloned DNA, inserts over 150,000 bp are very stable
YAC w/o telomere at the end are rapidly degraded

109
Q

cloned genes can be expressed to amplify protein production, and purified proteins have many purposes:

A

-clarify a proteins function
-study rxn mechanisms
-generate antibodies to the proteins
-reconstitute complex cellular activites in the test tube with purified components
-examining protein binding partners

110
Q

expression vectors

A

cloning vectors with transcription and translation signals needed for the regulated expression of a cloned gene
(necessary for expressing eukaryotic protein in bacteria, sequences dont function in bacteria)

111
Q

Any organism can serve as a host to express recombinant proteins:

A

-bacteria
-yeast
-insects
-mammalian cells in culture

112
Q

what is most common host for protein expression

A

bacteria

113
Q

bacterial hosts advantages

A

-regulatory sequences are well understood
-expresses high levels of cloned proteins
-easy to store and grow huge amount
-efficient methods for transforming and extracting DNA

114
Q

bacterial hosts disadvantage

A

-some heterologous proteins dont fold correctly

-proteins might not undergo the right posttranslational mods/proteolytic cleavage

-some gene sequences can be difficlut to express as eukaryotic proteins can aggregate into inclusion bodies

115
Q

transcription from the T7 promoter and RNA poly

A

-the cloned gene is fused to T7 promoter and transcribed by T7 RNA poly
-enables tight regulation

116
Q

yeast- what is the most understood eukaryotic organism

A

saccharomyces cervisiae

117
Q

principles of protein expression in yeast (same as bacteria)

A

-cloned genes must be linked to the appropriate promoter
-gene expression can be controlled by choosing an appropriate medium

118
Q

advantages of Yeast hosts

A

-well understood eukaryotic org
-expression of euk genes can be more efficient
-proteins can be folded and modded more accurately

119
Q

disadvantages of Yeast hosts

A

-heterologous proteins may not fold properly

-yeast may lack the enzymes needed to modify the proteins to their active forms

-certain features of the gene seq may stop expression of a protein

120
Q

Mammalian culturing
DNA introduced into mammalian cells (using engineered mammalian viruses as vectors)
advantages and disadvantages:

A

-proteins can be expressed either transiently or permanantly
-proper posttranslational mods can be ensured

disadvantages:
-can be very expensive

121
Q

site directed mutagenesis

A

technique used to individually replace specific AA’s

122
Q

oligonucleotide directed mutagenesis

A

technique used to create a specific DNA sequence change

123
Q

mutagenesis of the bacterial recA gene

A

Lys72 of RecA protein hydrolyzes ATP
-K72R mutant binds but doesnt hydrolyze ATP

124
Q

deletions

A

done by cutting out a segment with restriction endonucleases and ligating the remaining portions

125
Q

fusion protein

A

product of a ligated gene with parts of 2 different genes

126
Q

terminal tags provide handles for affinity purification. explain tags

A

-protein or peptide that binds a stable ligand with high affinity and specificity
-the tag is fused to gene encoding target protein
-permits purification by way of affinity chromatography

127
Q

tagged proteins in protein purification

A

provides good yield and high purity
may affect the properties of attached proteins

128
Q

RT-PCR

A

uses reverse transcriptase to generate a DNA strand from an RNA template. followed by standard PCR using DNA poly

129
Q

quantitative PCR qPCR or real time PCR

A

used to estimate relative copy numbers of particular sequences in a sample

130
Q

DNA library

A

collection of clones.
used for gene discovery and determination of gene/protein function

131
Q

cDNA (complementary DNA)

A

ds DNA fragments formed from mRNA templates, this relies on reverse transcriptase

132
Q

cDNA library

A

population of clones created by putting cDNA fragments into vectors and cloning

133
Q

gene library/combinatorial library

A

library focusing on sequence variants within one gene

134
Q

describe the 3 levels of protein function

A

phenotypic function- effects of a protein on the entire organism

cellular function- network of interactions a protein engages in at the cell level

molecular function- biochemical activity of a protein

135
Q

transcriptome

A

entire complement of transcribed RNAs present in the cell

136
Q

comparative genomics and genome annotation

A

cg= gene functions can be assigned by using genome databases to compare genes

genome annotation= converts seq of residues into useful info about the location/function of genes

137
Q

orthologs and paralogs

A

ortho- genes that occur in different species but have clear functional relationship to e/o

para- genes similarly related to e/o WITHIN a single species

138
Q

synteny

A

conserved gene order

-provides evidence for orthologous relationship between genes at identical locations within the related segments

139
Q

structural motifs

A

may help to define molecular function
certain AA seq associate w/ structural motifs
ex: hydrogenase

140
Q

mass spec can…

A

catalog and quanitfy thousands of proteins present in a cell
-complementary approach to RNA-seq
-provides info about how proteins are modded

141
Q

green fluorescent protein

A

jellyfish protein, serves as a useful location marker
-target gene fused to the GFP gene makes a highly fluorescent fusion protein

142
Q

immunoflourescence

A

alt approach to visualize the endogenous protein that involves fixation/death of the cell

143
Q

epitope tag

A

short protein seq, tightly bounded by an antibody

144
Q

knowing what a protein interacts with can suggest function.
techniques?

A

-purification of protein complexes
-yeast 2 hybrid analysis

145
Q

when and where a protein is present can suggest protein function
techniques?

A

-rna seq and transcriptomics
-cell proteomes/mass spec
-fusion proteins, and immunofluroescence

146
Q

immunoprecipitation

A

precipitating a fusion protein (that has the gene of interest and a gene for an epitope tag) by antibodies to the epitope

147
Q

tandem affinity purification - TAP tags

A

two consecutive tags that are fused to a target protein to enhance the selctivity of immunoprecipitation
-first tag (protein A) binds igG
-second tag (calmodulin binding peptide)- binds calmodulin

148
Q

yeast two hybrid analysis

A

relies on properties of the Gal4 protein
-the two domains of Gal4 must be brought together to function correctly
-probes molecular interactions in vivo

149
Q

the effect of deleting or altering a protein can suggest its function

A

CRISPR/cas used

150
Q

what does crispr stand for

A

clustered regularly interspaced short palindromic repeats

151
Q

CRISPR seqs

A

repeats in the bacterial genome surrounding sequences derived from phage pathogens that previously infected the bacterium

152
Q

cas protein is a…

A

nuclease

153
Q

components of the CRISPR/cas complex

A

guide RNAs- transcribed viral spaced sequences that are cleaved

trans-activating CRISPR RNA (tracrRNA)

1+ cas proteins

154
Q

the CRISPR/cas complex..

A

binds and destroys invading bacteriophage DNA by the Cas activities

155
Q

current CRISPR tech requires only 2 components

A

-single Cas9 protein
-single guide RNA - sgRNA

156
Q

what is sgRNA

A

single guide
made of gRNA and tracrRNA fused into a single RNA

157
Q

cas9 has two seperate nuclease domains

A

each domain cleaves one strand of DNA

158
Q

the sgRNA guide seq can be altered to..

A

target any genomic sequence

159
Q

_______ is required to pair with target DNA and activate the nuclease domains

A

cas9

160
Q

the operator of a e coli expression vector

A

permits regulation by a repressor that binds to it

Biochem (8 decks)

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