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+ BIOL 112 > FINAL Unit 2.5 > Flashcards

FINAL Unit 2.5 Flashcards

1
Q

What is a point mutation?

A

a different nucleotide is substituted

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2
Q

What is a polymorphism?

A

common genetic difference in a population

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3
Q

What is a single nucleotide polymorphism?

A

difference between two sequences at a single nucleotide position in DNA sequence

result of point mutation that occured in past and increased its frequency

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4
Q

What are the consequences of point mutations?

A
  • synonymous (silent): does not change amino acid (redundant, wobble)
  • nonsynonymous (missense): causes amino acid replacement
  • nonsense: creates stop codon that terminates translation. truncated polypeptides are almost always nonfunctional and quickly destroyed (eukaryotes can destroy mRNA with these codons)
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5
Q

What is a frameshift mutation?

A

inserting/deleting one or more nucleotides that changes the sequence of codons

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6
Q

What are the consequences of frameshift mutations?

A

mutant does not fold properly into its tertiary structure and is therefore nonfunctional

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7
Q

Predict the effects of mutations in coding vs. noncoding regions of the genome and when a mutation is inherited.

A
  • mutation in 5’ UTR of mRNA could affect translation because it is where RBS is
  • frameshift mutations have no affect in noncoding DNA
  • majority of DNA in genome doesn’t code for a protein, most sequences in noncoding DNA have no known function which explains why many point mutations in noncoding DNA have no detectable effect on the organism
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8
Q

What is CRISPR?

A

clustered regularly interspaced short palindromic repeats (gene editing)

describes the organization of viral DNA segments in bacterial genome

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9
Q

What happens when a bacterium is infected by a virus for the first time?

A
  • it makes a copy of part of the viral genome
  • on subsequent infection by the same virus, DNA copy of viral genome is transcribed to RNA that combines with protein that has DNA-cleaving function
  • RNA is guide to identify target DNA in virus by complementary base pairing and the protein cleaves the target DMA, ending the viral threat
  • bacteria remember and defend themselves from past infections
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10
Q

What is the role of CRISPR and nuclease Cas 9 in bacterial defence against viruses?

A
  1. CRISPR arrays have pieces of viral genomes inserted into bacterial DNA
  2. in CRISPT arrays, spacer virus sequences are separated by identical ‘repeat’ sequences
  3. CRISPR RNA sequences bind to an enzyme called Cas9, transcribing CRISPR sequences produces RNA sequences (crRNA)
  4. bacteria is ready to defend itself from new virus: CRISPR-Cas9 complex uses crRNA to find a sequence math in virus genome and Cas9 opens DNA and cuts virus genome
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11
Q

What is required for CRISPR/Cas9 genome editing?

A
  • guide RNA: engineered to be complementary to target DNA
  • Cas9: a gene for Cas9 cleaves DNA when it associates with guide RNA
  • DNA that acts as template: piece with desired new sequence for target DNA

they all contain target DNA for sequence wanted to be edited

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12
Q

Describe the process of CRISPR genome editing.

A

overall: target DNA identified by guide RNA, cleaved by Cas9, and replaced with sequence of template DNA

  1. transform cell with plasmid that contains sequences that code for CRISPR guide RNA and Cas9
  2. guide RNA undergoes base pairing with target DNA
  3. Cas9 cleaves target DNA
  4. exonucleases in cell expand gap, gap is repaired using new template DNA for editing target DNA
  5. strands of gapped target DNA undergo base pairing with complementary ends of editing template
  6. DNA synthesis elongates target DNA strands and closes gap

result: target DNA restored, but sequence is altered according to editing template

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13
Q

What is transcriptional regulation?

A
  • how frequently a gene is transcribed

- changes how easy it is to recruit RNA POL to promoter

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14
Q

What is translational regulation?

A
  • how frequently mRNA is translated
  • change rate mRNA is degraded
  • change rate of translation by ribosomes
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15
Q

What is post-translational regulation?

A
  • how frequently a protein functions
  • changes protein shape by binding activator/inhibitor
  • changes protein shape by chemical modification
  • change rate protein is degraded
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16
Q

What is a constitutively expressed gene?

A

gene that is expressed all the time because its gene product is needed all the time (ie. rRNA, tRNA, RNA POL, ribosomal proteins, amino acyl tRNA synthetase)

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17
Q

What is an environmentally-regulated gene?

A

gene whose expression level is linked to a condition in the environment such as nutrient availability (ie. mal and lac operons)

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18
Q

What is a developmentally-regulated gene?

A

expressed only at specific developmental periods of an organism

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19
Q

What is basal level?

A

low amount of transcription

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20
Q

What is the regulation of gene expression critical for?

A

efficient use of resources and therefore, survival

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21
Q

How do prokaryotes regulate genes?

A

in clusters called operons

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22
Q

What is an operon?

A

set of coding sequences for related proteins all sharing the same promoter and terminator, and contains an operator

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23
Q

What is an operator?

A

region of DNA where regulatory proteins bind, sometimes overlaps with promoter

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24
Q

What does the transcription of an operon result in?

A

one long mRNA encoding multiple proteins called polycistronic mRNA

  • each section of mRNA encoding one polypeptide must have an upstream RBS
  • RNA POL transcribes polycistronic RNA
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25
Q

Contrast transcription of operon with transcription in eukaryotes.

A

eukaryotes: one gene encodes one pre-mRNA
- each mRNA encoding one polypeptide has 5’ cap/5’ UTR for ribosome binding
- promoter controls gene expression by binding transcription factors
- each gene has its own promoter
- RNA POL transcribes monocistronic mRNA

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26
Q

Compare the general mechanisms of positive and negative regulation.

A

positive:

  • activator protein binds
  • RNA POL binds
  • transcription occurs

negative:

  • repressor protein binds
  • no RNA POL binds
  • no transcription occurs
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27
Q

What does derepressed mean?

A

if the repressor isn’t present in negative transcriptional regulation, native state of DNA allows RNA POL to be recruited and transcription occurs

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28
Q

What is a weak promoter?

A

if gene has an activator, its promoter must not strongly recruit RNA POL on its own

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29
Q

What is a strong promoter?

A

gene with repressor can recruit RNA POL without help

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30
Q

What is an inducer?

A

small molecule that interacts with repressor and prevents it from biding DNA and blocking transcription (ie. lactose and maltose), triggers operon expression

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31
Q

Describe lac operon.

A

negative regulation, cells import lactose into cell and break it down for energy

32
Q

What is lacY?

A

gene that codes for protein lactose permeate that transports lactose from outside to inside the cell

33
Q

What is lacZ?

A

gene that codes for enzyme beta-galactosidase that cleaves lactose molecule into glucose and galactose constituents

34
Q

What is the regulation of lacY and lacZ controlled by?

A

product of lacI which encodes the repressor and is constitutively expressed

35
Q

Bacteria regulate enzymes to break down lactose using the lac operon. What happens in the absence and presence of lactose?

A

absence: repressor binds to operator and prevents transcription
presence: repressor is unable to bind to operator, promoter recruits RNA POL complex and transcription of polycistronic mRNA occurs

36
Q

How does the presence of lactose result in the induction of the lac operon?

A

low lactose: LacI binds operator region of DNA near promoter to negatively regulate transcription, lac operon is repressed by LacI but small amount of transcription can occur

no lactose: no need to break down enzymes

high lactose: negative regulation is removed so operon is transcribed
- LacI binds lactose, released from operator, operon transcribed

37
Q

What happens when LacI is bound to lactose?

A
  • LacI changes tertiary structure and cannot bind to operator
  • RNA POL can be recruited to promoter
  • lac operon is transcribed at high levels
  • LacZ, LacY, LacA proteins are translated
38
Q

What is mal operon?

A

positive regulation

  • MalT activates transcription by helping RNA POL to bind
  • if MalT is removed, RNA POL cannot bind and no transcription occurs
39
Q

Regulation isn’t absolutely an ON/OFF thing, it’s more about an increase/decrease in gene expression.

A
  • LacI binds to operator, but there’s an equilibrium, a t some point the repressor releases from operator even when lactose is absent
  • during the brief time when LacI isn’t bound to DNA, RNA POL can transcribe operon at basal level
  • in low lactose, LacI has high affinity for DNA so it’s usually, but not always, bound to DNA: gene expression at basal level
  • deletion of promoter for lac operon could make gene expression levels lower than basal
40
Q

What happens when maltose is low?

A

mal operon is off but basal level of transcription still occurs

41
Q

What happen when maltose is present/high?

A

mal operon is on

  1. MalT binds maltose
  2. MalT changes shape and has higher affinity for operator
  3. MalT binds operator, RNA POL can bind more effectively to malPQ promoter
  4. RNA POL is recruited (strong promoter)
  5. T&T of MalP and malQ occurs in high levels
  6. MalP and MalQ break down maltose
42
Q

What happens when maltose is absent?

A

little transcription

  • low amounts of MalT always made
  • no maltose for binding, therefore does not bind to operator
  • RNA POL doesn’t bind to promoter strongly (weak promoter) and little transcription of malPQ operon occurs
  • low/no production of MalP and MalQ enzymes
  • MalT on its own is a very weak DNA binding protein
43
Q

How is maltose being consumed by the cell?

A
  • like all binding event in cell, binding of maltose to MalT isn’t permanent
  • concentration of maltose decreases, maltose is released from MalT and MalT returns to original shape with low affinity for operator and transcription is no longer activated
  • levels of MalP and MalQ in cell dilute as cell continues to grow and divide
44
Q

What is polymerase chain reaction?

A

DNA synthesis in vitro = in a test tube

method for making copies of a piece of DNA which allows a targeted region of a DNA molecule to be replicated (amplified) into any amount of copies over and over

45
Q

What are the 3 steps of PCR?

A
  1. denaturation: heating solution in plastic tube to a temperature just short of boiling so that the individual DNA strands of the template separate (H bonds are broken)
  2. annealing: (begins as solution is cooled) because of great excess of primer molecules, 2 primers bind (anneal) to their complementary sequence on DNA (rather than the two strands of template duplex coming back together)
  3. extension: solution is heated to optimal temperature for Taq DNA POL and the polymerase elongates each primer with deoxynucleoside triphosphates to synthesize new DNA strands

after sufficient time to allow new DNA synthesis, solution is heated again and the cycle repeats

46
Q

What are the 4 components needed for PCR?

A
  • template DNA
  • DNA primers (2)
  • Taq DNA POL
  • all 4 deoxynucleoside triphosphates
47
Q

Describe template DNA required for PCR.

A
  • at least one molecule of double-stranded DNA containing the region to be amplified serves as a template for amplification
  • usually a sequence of double-stranded DNA larger than gene(s) of interest, often genomic DNA
48
Q

Describe DNA primers (2) required for PCR.

A
  • two short sequences of single-stranded DNA are required for Taq DNA POL to start synthesis
  • enough primer added so number of primer DNA molecules is much greater than number of template DNA molecules
  • determines what sequence of DNA will be amplified
  • are oglionucleotides (produced by chemical synthesis, 20-30 nucleotides long and sequence is complementary to ends of the region of DNA to be amplified
  • 3’ end must be oriented towards region to be amplified so when Taq DNA POL extends it, it creates a new DNA strand complementary to targeted region
  • one primer pairs with one strand, the other primer pairs with other strand
  • Taq DNA POL adds new nucleotide to 3’ OH group on primer
49
Q

Describe Taq DNA POL required for PCR.

A

(special DNA POL for PCR)

carries out template-directed synthesis, temperature optimum is 75-85 degrees C ideal for PCR

50
Q

Describe the repeated cycle of amplification.

A
  • in each cycle, number of copies of targeted fragment is doubled (2^n, n = number of rounds)
  • by the third round, process begins to produce molecules that are only as long as region of the template duplex flanked by sequences complementary to primers
  • DNA POL loses structure and function every round therefore, new DNA POL is needed for each cycle: Taq DNA POL is used instead of DNA POL
51
Q

When does DNA replication happen in cells?

A

prokaryotes: during growth, before cell divides
eukaryotes: during interphase to make 2 copies of the genome, which get divided by mitosis

52
Q

What happens after two rounds of DNA replication?

A

one daughter is half-labeled, one daughter is fully-labeled

53
Q

What is DNA replication?

A

DNA replication in vivo = inside an organism

54
Q

What is the function of DNA replication?

A

daughter cells will need a copy of the parent genome

55
Q

What is the DNA replication fork and how does it work?

A

where parental strands separate and new DNA grow by addition of nucleotides to the 3’ end

  • moves forwards as more and more parental DNA is unwound
  • one strand grows in the opposite direction of the fork because nucleotides
  • DNA polymerization occurs only in 5’ to 3’ direction because of chemistry of nucleic acid synthesis
56
Q

What are the steps of DNA replication?

A
  1. strands unwind: results in a replication fork with leading and lagging strands
  2. RNA primase makes RNA primers for DNA POL to extend
    - RNA primase lays down an RNA primer
  3. DNA POL adds NTP to the polymer
    - DNA POL extends the RNA primer by adding nucleotides to 3’ end, and all new DNA strands have short RNA segment at 5’ end
    - incoming nucleotides are accepted if they correctly base pair with template
    - 3’ OH of growing strand attacks high-energy phosphate bond of incoming nucleotide to initiate synthesis reaction
  4. a different DNA POL removes primer when growing fragment meets fragment before it and replaces it with DNA (nucleotides to fill space of removal), and DNA ligase forms a bond joining the two DNA fragments
57
Q

What is a leading strand?

A

continuous strand

as helix unwinds, nucleotides can be added to 3’ end and this daughter strand can be synthesized as one long continuous polymer

58
Q

What is a lagging strand?

A

5’ end is oriented towards replication fork but cam’t grow in that direction so instead, when the fork unwinds, it grows away

  • as parental helix unwinds, new daughter strand is initiated with its 5’ end near fork, and strand is elongated at 3’ end
  • results in okazaki fragments: short, synthesized pieces that are elongated at 3’ end until it reaches the piece in front of it
59
Q

What accessory proteins get DNA ready for replication?

A

helicase: unwinds parental double helix
topoisomerase: relieves the stress of unwinding

single-stranded binding proteins: stabilize large strands of DNA

60
Q

Describe the function of primers in DNA replication.

A

primer: short stretch of that starts DNA synthesis
- needed because DNA POL complex can’t begin new strand on its own, it can only elongate the end of an existing piece of DNA or RNA
- made by RNA primase

61
Q

What is RNA primase?

A

RNA POL that synthesizes a short piece of RNA complementary to DNA template

62
Q

What is the proof-reading function?

A

most DNA can correct their own errors during replication

  • when each new nucleotide comes into line to attach to growing strand, it is held temporarily by H bonds for complementary base pairing
  • when an incorrect nucleotide is attached to new DNA strand, DNA POL can correct it because it detects mispairing between template and most recently added nucleotide
  • this activates the DNA-cleavage function of DNA POL that removes the incorrect nucleotide and inserts the correct one
63
Q

What is exonuclease activity?

A

DNA POL can back up a few bases and cut out incorrect nucleotide and replace it with correct one

64
Q

What are the mechanisms of DNA repair after DNA replication occurs?

A
  1. mismatch repair

2. DNA ligase repair

65
Q

What is mismatch repair?

A
  • mismatch detected by proteins
  • exonuclease removes bases
  • DNA POL fills in missing nucleotides
  • DNA ligase joins the backbones
66
Q

What is DNA ligase repair?

A
  • mutagens can damage DNA backbone by causing break in one strand
  • DNA ligase can reseal backbone by joining 3’ and 5’ ends together (catalyze a phosphodiester bond)
  • cells can have different types of DNA ligase
  • another type of DNA ligase repairs double-stranded breaks
67
Q

What is the origin of replication for chromosome replication?

A

each point at which DNA synthesis is initiated

68
Q

What is the origin of replication chromosome replication in bacteria?

A

one origin

replication starts at origin and moves around the circular chromosome in both directions (takes place at both forks)

69
Q

What is the origin of replication chromosome replication in eukaryotes?

A

many origins

  • replication can begin at any origin
  • each replication bubble formed by the opening of double helix at each origin, has two replication forks that move in opposite directions
  • replication bubble grows as replication continues
  • DNA synthesis takes place at each fork: new strand with free 3’ end is leading strand and that with 5’ free end is lagging strand
  • when two replication bubbles meet, they fuse to make one large bubble
70
Q

What does telomerase do?

A

restores tips of chromosomes shortened during DNA replication

71
Q

Describe the replication of circular DNA.

A

circular DNA can be replicated completely: no ends, full circles

72
Q

Describe the replication of linear DNA.

A

linear DNA have ends, at each round of replication the ends are slightly shorter

  1. leading strand replicates whole strand
  2. last RNA primer on lagging strand sits near end of template
  3. RNA primer is removed, section of template DNA remains unreplicated
  4. in next round, shortened template results in shorter chromosome
  5. if the pattern persists, chromosomes will get shorter and shorter
73
Q

What is a telomere?

A

repeating sequence at each end of a eukaryotic chromosome that differs from one group of organisms to another

  • slightly shortened in each round of DNA replication, but its quickly restored by enzyme telomerase which contains an RNA molecule complementary to telomere sequence
74
Q

How does telomerase restore telomere?

A
  1. terminal end of telomere in template remains unreplicated
  2. telomerase restores 3’ shortened end
  3. new segments of lagging strand can be formed so original telomere in template is completely restored
  4. human chromosome contains thousands of telomere repeats
75
Q

What are the key properties of DNA POL?

A
  • reads sequence on template and links nucleotides together
  • cannot start on its own therefore must always start from existing 3’ OH end of an existing template (ie. primer)

except in the cell, the primer is RNA not DNA

76
Q

Describe DNA interactions with proteins.

A
  • DNA binding proteins must interact with DNA for DNA replication (ie. primase must bind to DNA strand to read template and synthesize RNA primer, DNA POL must bind to DNA strand to read template)
  • when each enzyme has completed its task, enzyme must be released from DNA (these non-covalent interactions are temporary)

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