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
2
Q
Functions of DNA
A
Storage of biological info
Transmission of that info to next gen
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)
4
Q
Three components of nucleotides
A
-nitrogenous base (pyramiding or purine)
-Pentose
- +1 phosphates
5
Q
What is a nucleoside
A
Molecule without phosphate
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
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
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)
9
Q
What are the 4 major deoxyribonucleotides (structural units of DNA)
A
-deoxyadenylate
-deoxyguanylate
-deoxythymidylate
-deoxycytidylate
10
Q
What are the 4 major ribonucleotides (structural units of RNA)
A
-adenylate
-guanylate
-uridylate
-cytidylate
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)
12
Q
Phosphodiester linkage
A
Covalent bond that joins successive nucleotides of both DNA and RNA
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 \
14
Q
What is oligonucleotide and polynucleotide
A
Oligo- short (<50 nt) nucleic acid
Poly- longer nucleic acid
15
Q
Describe nucleotide bases
A
Weakly basic compounds.
-aromatic
-pyrimidines are planar, purines have pucker
16
Q
Free pyrimidine and purine bases may exist as…
A
Tautomers
-lactam, lactim, double lactim
17
Q
Nucleotides and UV absorption
A
All nucleotide bases absorb UV light, strong at 260nm
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
19
Q
Describe base pairs
A
H bonding patterns between complementary strands of nucleic acids , A to T or U, and G to C
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
21
Q
What did x ray diffraction reveal
A
DNA molecules are helical
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
23
Q
Phosphodiester bonds can… (in DNA strands )
A
Run in the same or opposite directions- parallel or antiparallel
24
Q
Double helix has ____ bp per helical turn
A
10.5bp
25
Hydrogen bonding contributes to stability of DNA structure
Not really
26
Double helical DNA strands are…
Complementary
27
How is the DNA double helix stabilized
-metal cations that shield negative charges of backbone phosphates
-base stacking interactions between successive base pairs.
-duplexes with lots of GC are MORE stable
28
The 2 steps for replication of DNA
1. Parent strands become separated
2. Each parent strand serves as a template for synthesis of complementary daughter strand
29
Examples of structural variation in DNA
-different conformations of deoxyribose
-rotation about the phosphodeoxyribose backbone
-free rotation about the C-1’-N glycosyl bond
30
Describe the 3 forms of DNA
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
31
Palindrome DNA occurs in
Regions with inverted repeats
32
What is a mirror repeat
Occurs when the inverted repeat is in each individual strand
33
Hairpin and cruciform structures occur from…
The self complementarity in each strand
34
DNA structures with 3 strands
Hoogsteen positions (N7, O6,, and N6 of purines)
Hoogsteen pairing- non- WC pairing
Triplex DNAs- form from Hoogsteen pairing
35
DNA structures with 4 strands
Tetraplex DNA -4 DNA strands pair, only when there’s a high level of G residues
G tetraplex= very stable
36
Transcription is..
Process of mRNAs forming on a DNA template.
MRNA- part of RNA carrying genetic info from DNA to ribosome
37
MRNAs code for
Polypeptide chains
38
MRNA can be. Monocistronic or polycistronic - describe
Mono- codes for one polypeptide (most mRNA in eukaryotes)
Poly- codes for 2+ polypeptides, in bacteria and archaea
39
MRNA is always ______ stranded (describe)
Single.
-with right handed conformation, and base stacking interactions
-can base pair w complementary DNA or RNA (paired strands are antiparallel)
40
Describe secondary structure of RNA
-complementary RNA strands form an A form right handed double helix
-breaks result in bulges or internal loops
-internal loops form palindromic sequences
41
Base paired helical structures in RNA
-base paired helical segments form in RNA
-hair pins most common
42
The off set pairing in 3D DNA structure creates…
Major and minor grooves on the surface
43
Desaturation of double helical RNA/DNA occurs by…
-high temp or extreme change in pH
-will disrupt H bonds and base stacking interactions
44
Double helical DNA/RNA can anneal (explain )
Annealing- 2 strands spontaneously rewind when Temp orPH Is returned to normal range
45
Hypochromic effect
Observed decrease in absorption of UV light when complementary strands are paired
46
Hyperchromic effect
Increase in absorption of UV light when a double stranded nucleic acid is denatured
47
Heat desaturation of DNA
Temp where 1/2 of DNA is separated single strands
-this increases with GC pairs
48
Partially denatured DNA is rich with ____ bp’s
AT
49
____ duplexes are more stable to heat denaturation than ___ duplexes
RNA, DNA
50
Nucleotides and nucleic acids undergo mutations which are…
Nonenzymatic transformations
51
Deamination
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
52
Depurination
Hydrolysis of N-B-glycosyl bond between the base and Pentose , and creates an Apurinic site (AP)
53
Reactions made by radiation
UV light causes cyclobutane pyrimidine dimers
Ionizing radiation causes ring opening, base fragmentation, and breaks in covalent backbone of nucleic acids
54
DNA damage by reactive chemicals (give examples)
Nitrous acid precursors are deaminating agents
Alkylating agents = create modified nucleotides (non-enzymatically)
55
Alkylating agents can alter bases of DNA..
-they can methylate guanine to O6-methyl-guanine, which can’t base pair with cytosine
56
Explain DNA damage by oxidative damage
Reactive oxygen species (hydrogen peroxide, hydroxyl radicals, superoxide radicals) can damage DNA
-OH radicals are most common for oxidative DNA damage
57
Some bases of DNA are methylated…
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
58
Chemical synthesis of DNA by ________ method is highly efficient
Phosphoramidite
59
Describe PCR
-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
60
Sanger sequencing
Dideoxy chain termination sequencing, and ddNTPs interrupt synthesis
-each four of the ddNTPs are labeled with a fluorescent tag
61
Describe reversible terminator sequencing
Uses 4 different modified deoxynucleotides (A,T,C,G), each with a certain fluorescent label and 3’ blocking group
62
Sequencing depth
Number of times a particular nucleotide in a genome is sequenced
63
Contigs
Long, contiguous sequences that are assembled from overlaps
64
Hydrolysis of ________ provide chemical energy (ATP)
nucleoside triphosphates
65
When coupled to a ________ ATP hydrolysis shifts eq to product
Positive free energy change
66
Nucleotide binding fold
Single protein domain that binds adenosine
67
Second messengers
Compounds made in the cell following interaction of extracellular chemical signals with receptors (cAMP)
68
PpGpp
Made in bacteria during AA starvation to inhibit the synthesis of the rRNA and tRNA molecules
69
Adenine nucleotides can serve as signals
ATP and ADP:
-neurotransmitters in a variety of signaling pathways
-signals for receptors that mediate pain sensation
-blood clotting signals
70
Which nucleoside is commonly found as part of coenzymes
Adenosine
71
How nucleotides be involved in signaling
Precursors for making second messengers
Extracellular ADP and ATP binding to its receptor
72
Explain DNA Cloning
Selective amplification of a particular gene or DNA segment
73
Explain recombinant DNA tech (genetic engineering)
Methods to do DNA cloning
74
the 5 processes of DNA cloning
-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
what are cloning vectors and recombinant DNAs?
cloning vector- small DNA capable of autonomous replication
recombinant DNA- composite DNA made of covalently linked segments from 2 or more sources
76
what are the advantages of cloning in E coli?
-DNA metabolism is well understood
-many naturally occuring cloning vectors like plasmids and bacteriophages
-techniques for moving DNA from one bacteria to another
77
enzyme used in recombinant DNA tech:
Type 2 restriction endonuclease
cleaves DNA at specific base sequences
78
enzyme used in recombinant DNA tech:
DNA ligase
joins 2 DNA molecules or fragments
79
enzyme used in recombinant DNA tech:
DNA poly I
Fills gaps in duplexes by stepwise addition of nucleotides to 3' ends
80
enzyme used in recombinant DNA tech:
reverse transcriptase
makes a DNA copy of an RNA molecule
81
enzyme used in recombinant DNA tech:
polynucleotide kinase
adds phosphate to the 5' OH end of a polynucleotide to label it, or to help ligation
82
enzyme used in recombinant DNA tech:
terminal transferase
adds homopolymer tails to the 3' OH ends of a linear duplex
83
enzyme used in recombinant DNA tech:
exonuclease III
removes nucleotide residues from the 3' ends of a DNA strand
84
enzyme used in recombinant DNA tech:
bacteriophage λ exonuclease
removes nucleotides from the 5' ends of a duplex to expose single stranded 3' ends
85
enzyme used in recombinant DNA tech:
alkaline phosphotase
removes terminal phosphates from the 5' end and 3' end
86
restriction endonuclease AKA restriction enzymes
recognize and cleave DNA at specific sequences (recognition sites)
87
methylases
catalyze methylation of host DNA to protect it from digestion by the host cell's restriction endonucleases
88
restriction mod system
restriction endonuclease and the corresponding methylase
89
types of restriction endonucleases
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
size of type 2 endonucleases
4-6 bp long
palindromic
91
DNA ligase
joins DNA fragment to be cloned into a suitable cloning vector
92
restriction endonucleases can leave either sticky ends or blunt ends (describe)
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
the DNA segment to be cloned is made by PCR, and including the _______ facilitates the cloning of amplified DNA
restriction endonuclease cleavage sites
94
cleavage of PCR amplified DNA creates sticky ends used to...
ligate the amplified DNA to a cloning vector
95
describe linkers and multiple cloning site
linker- synthetic DNA fragments created to bridge ligated ends
multiple cloning site- inserted DNA fragment that has multiple recognition sequences for restriction endonucleases
96
what are the three cloning vectors
-plasmids
-bacterial artificial chromosomes
-yeast artificial chromosomes
97
describe plasmids
circular DNA
-replicates seperately from the host chromosome
-5,000 to 400,000 bp
-symbiotic role in cell
98
features of the E coli plasmid
-origin of replication: where replication is initiated
-resistance genes
-recognition sequences for restriction endonucleases
99
describe bacterial transformation and electroporation
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
selectable markers and screenable markers are used to identify cells that take up plasmid DNA
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
plasmid pBR322 makes __________ selection marker
positive and negative
102
shuttle vectors
plasmids that can be propagated in cells of 2+ species , can incorporate multiple replication origins
103
plasmid vectors allow for cloning of very long DNA
100,000 to 300,000 bp
104
BACs are made up of...
made up of plasmid vector and large segments of cloned DNA
105
features of the BAC vector
- 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
what is the YAC made up of...
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
the YAC vector has elements to maintain a eukaryotic chromosome in yeast nucleus:
-yeast ORI
-2 selectable markers
-specialized sequences for stability and chromosome segregation at cell division- the centromere and 2 telomeres
108
stability of YAC clones...
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
cloned genes can be expressed to amplify protein production, and purified proteins have many purposes:
-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
expression vectors
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
Any organism can serve as a host to express recombinant proteins:
-bacteria
-yeast
-insects
-mammalian cells in culture
112
what is most common host for protein expression
bacteria
113
bacterial hosts advantages
-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
bacterial hosts disadvantage
-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
transcription from the T7 promoter and RNA poly
-the cloned gene is fused to T7 promoter and transcribed by T7 RNA poly
-enables tight regulation
116
yeast- what is the most understood eukaryotic organism
saccharomyces cervisiae
117
principles of protein expression in yeast (same as bacteria)
-cloned genes must be linked to the appropriate promoter
-gene expression can be controlled by choosing an appropriate medium
118
advantages of Yeast hosts
-well understood eukaryotic org
-expression of euk genes can be more efficient
-proteins can be folded and modded more accurately
119
disadvantages of Yeast hosts
-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
Mammalian culturing
DNA introduced into mammalian cells (using engineered mammalian viruses as vectors)
advantages and disadvantages:
-proteins can be expressed either transiently or permanantly
-proper posttranslational mods can be ensured
disadvantages:
-can be very expensive
121
site directed mutagenesis
technique used to individually replace specific AA's
122
oligonucleotide directed mutagenesis
technique used to create a specific DNA sequence change
123
mutagenesis of the bacterial recA gene
Lys72 of RecA protein hydrolyzes ATP
-K72R mutant binds but doesnt hydrolyze ATP
124
deletions
done by cutting out a segment with restriction endonucleases and ligating the remaining portions
125
fusion protein
product of a ligated gene with parts of 2 different genes
126
terminal tags provide handles for affinity purification. explain tags
-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
tagged proteins in protein purification
provides good yield and high purity
may affect the properties of attached proteins
128
RT-PCR
uses reverse transcriptase to generate a DNA strand from an RNA template. followed by standard PCR using DNA poly
129
quantitative PCR qPCR or real time PCR
used to estimate relative copy numbers of particular sequences in a sample
130
DNA library
collection of clones.
used for gene discovery and determination of gene/protein function
131
cDNA (complementary DNA)
ds DNA fragments formed from mRNA templates, this relies on reverse transcriptase
132
cDNA library
population of clones created by putting cDNA fragments into vectors and cloning
133
gene library/combinatorial library
library focusing on sequence variants within one gene
134
describe the 3 levels of protein function
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
transcriptome
entire complement of transcribed RNAs present in the cell
136
comparative genomics and genome annotation
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
orthologs and paralogs
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
synteny
conserved gene order
-provides evidence for orthologous relationship between genes at identical locations within the related segments
139
structural motifs
may help to define molecular function
certain AA seq associate w/ structural motifs
ex: hydrogenase
140
mass spec can...
catalog and quanitfy thousands of proteins present in a cell
-complementary approach to RNA-seq
-provides info about how proteins are modded
141
green fluorescent protein
jellyfish protein, serves as a useful location marker
-target gene fused to the GFP gene makes a highly fluorescent fusion protein
142
immunoflourescence
alt approach to visualize the endogenous protein that involves fixation/death of the cell
143
epitope tag
short protein seq, tightly bounded by an antibody
144
knowing what a protein interacts with can suggest function.
techniques?
-purification of protein complexes
-yeast 2 hybrid analysis
145
when and where a protein is present can suggest protein function
techniques?
-rna seq and transcriptomics
-cell proteomes/mass spec
-fusion proteins, and immunofluroescence
146
immunoprecipitation
precipitating a fusion protein (that has the gene of interest and a gene for an epitope tag) by antibodies to the epitope
147
tandem affinity purification - TAP tags
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
yeast two hybrid analysis
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
the effect of deleting or altering a protein can suggest its function
CRISPR/cas used
150
what does crispr stand for
clustered regularly interspaced short palindromic repeats
151
CRISPR seqs
repeats in the bacterial genome surrounding sequences derived from phage pathogens that previously infected the bacterium
152
cas protein is a...
nuclease
153
components of the CRISPR/cas complex
guide RNAs- transcribed viral spaced sequences that are cleaved
trans-activating CRISPR RNA (tracrRNA)
1+ cas proteins
154
the CRISPR/cas complex..
binds and destroys invading bacteriophage DNA by the Cas activities
155
current CRISPR tech requires only 2 components
-single Cas9 protein
-single guide RNA - sgRNA
156
what is sgRNA
single guide
made of gRNA and tracrRNA fused into a single RNA
157
cas9 has two seperate nuclease domains
each domain cleaves one strand of DNA
158
the sgRNA guide seq can be altered to..
target any genomic sequence
159
_______ is required to pair with target DNA and activate the nuclease domains
cas9
160
the operator of a e coli expression vector
permits regulation by a repressor that binds to it
