Molecular Biology Exam I

Front 1

When did Molecular Biology begin?

Back 1

Mid 1800's

Front 2

Define Molecular biology

Back 2

the study of the reproduction and expression of genetic information

Front 3

When did Beadle and Tatum propose the one gene-one enzyme hypothesis?

Back 3

1941

Front 4

Define the RNA world hypothesis and its corollary

Back 4

Posits that there was a stage early in the evolution of life that was dominated by RNA. Corollary: The current functions of RNA are only remnants of its previous functions

Front 5

What evidence supports the RNA world hypothesis?

Back 5

1. Self-splicing introns
2. RNAse P
3. Self-cleaving viral RNAs
4. Peptidyl transferase in the ribosome
5. Ribonucleotides are involved in metabolism, signalling, assembly of complexes, and energy for motility, ion pumping, etc

Front 6

Describe the structure of 2-deoxy-adenosine

Back 6

Purine (2 rings) and NH2

Front 7

Describe structure of 2-deoxy-guanosine

Back 7

Purine and O

Front 8

Describe structure of 2-deoxy-guanosine

Back 8

Pyrimidine and O

Front 9

Describe structure of 2-deoxy-cytidine

Back 9

pyrimidine and NH2

Front 10

What are the molecular differences between RNA and DNA?

Back 10

2'hydroxyl group in RNA, uracil vs thymine (CH3 at C1)

Front 11

What holds nucleotides together in DNA and RNA?

Back 11

phosphodiester bonds

Front 12

What are stem loops?

Back 12

Double-stranded regions of RNA that are A form helices with SS loop at end

Front 13

What chemical forces hold DNA or RNA strands together?

Back 13

1. H bonds
2. Electrostatic forces
3. Van der Waals interactions

Front 14

What are the characteristics of B form DNA?

Back 14

1. major groove and minor groove
2. 10 bps per turn
3. 3.4 nm turn
4. right handed

Front 15

What graph shows helix stability?

Back 15

Melting curves

Front 16

Describe the experiment that found the minimum origin length

Back 16

starting with e.coli chromosome and plasmid, cut each with a restriction enzyme and combine the fragments. use ampicillin to narrow down recombinant DNA with functional origin and amp gene. Repeat.

Front 17

What does the OriC consensus sequence consist of?

Back 17

3 13-mers, a 9a-mer, 3 9b-mers

Front 18

What is the minimum origin sequence?

Back 18

245bp

Front 19

What events occur to open the origin?

Back 19

1. DnaA's bind 9-mers, and with help of Hu and ATP, DNA wraps around
2. 13-mers melt, forming Open Complex
3. DnaB, with the help of DnaC and ATP, binds, forming prepriming complex
4. Primase binds DnaB and DNA, then this complex moves along template and makes RNA primers

Front 20

What enzyme is DnaB?

Back 20

helicase

Front 21

List the enzymes involved in DNA elongation

Back 21

1. DNA-dependent DNA polymerases
2. Primase
3. DNA ligase
4. Helicase
5. SSB
6. Gyrase

Front 22

What is the mechanism of Prokaryotic DNA ligase?

Back 22

Ligase binds NAD, cleaves it, Ligase-AMP binds to 5' end of DNA, 3' end reacts with phosphodiester bond, displacing Ligase-AMP

Front 23

What are the functions of SSB?

Back 23

1. prevents strands from reannealing
2. protects strands from degradation
3. stimulates DNA Pol III and helicase

Front 24

How many subunits do DNA Pol I and DNA Pol III have?

Back 24

DNA Pol I = 1
DNA Pol III = 10

Front 25

Name the major subunits of DNA Pol III and their functions

Back 25

1. alpha - DNA synthesis
2. Epsilon - 3'-5' exonuclease
3. Theta - stimulate Epsilon
4. Beta clamp - improve processivity
5. Gamma complex - loads Beta clamp
6. Tau - holds the two cores together

Front 26

What experiment was used to show that the B clamp slides like a donut on DNA?

Back 26

size exclusion chromatography with circular DNA and linear DNA. control gel with ligase.

Front 27

How is DNA synthesis terminated in prokaryotes?

Back 27

Each fork goes past one Ter region, stops at the next, and binds the tus protein

Front 28

What performs decatenation in E. coli?

Back 28

Topoisomerase IV (a type II)

Front 29

What are the 5 eukaryotic DNA polymerases? give their functions

Back 29

alpha - primase
delta - elongates primers, highly processive, proofreading
epsilon and beta - DNA repair
gamma - replication of mitochonrial and chloroplast DNA

Front 30

What enzyme in eukaryotes has 5' to 3' exonuclease activity, removing RNA primers?

Back 30

FEN-1

Front 31

What is the eukaryotic equivalent to the Beta clamp and SSB?

Back 31

PCNA and RP-A

Front 32

Describe MtDNA

Back 32

1. multi-copy, circular, 16,000 bp
2. encodes genes for respiration and translation
3. 2 promoters
4. mutations in MtDNA can cause genetic diseases

Front 33

How is mtDNA replicated?

Back 33

1. replication begins on heavy strand from Oh, once it reaches Ol, replication of the light strand begins in opposite direction
2. Lagging L strand replication gives alpha and beta molecules.
3. beta molecule finishes replication
4. both alpha and beta supercoil

Front 34

what is the centiMorgan?

Back 34

unit of recombination frequency, represents 1% recombination frequency

Front 35

What are the three types of recombination?

Back 35

1. Homologous
2. Site Specific
3. Transposition

Front 36

What are the steps in the recBCD pathway of homologous recombination?

Back 36

1. recBCD makes nick in one strand at Chi sequence
2. recBCD unwinds DNA w/ Chi sequence, SSB and rec A bind
3. recA promotes strand invasion
4. displaced strand pairs with SS left behind
5. recBCD nicks displaced strand
6. nicks are sealed (Holliday juction)
7. ruvA and ruvB promote branch migration
8. ruvC resolves holliday juction

Front 37

What are the enzymatic activities of RecBCD?

Back 37

1. endonuclease (recBCD)
2. DNA helicase (recBC)
3. DNA-dependent ATPase

Front 38

What is RecA?

Back 38

38 kDa protein, polymerizes onto SS DNA 5'-3'(binds DS DNA weakly), catalyzes strand exchange, ATPase

Front 39

In what steps does Rec A function?

Back 39

Pre-synapsis, synapsis, and post-synapsis (strand exchange)

Front 40

What do RuvA and Ruv B do?

Back 40

RuvA binds the HJ and 2 RuvB(helicase + ATPase) bind to ruvA and 2 DNA helicases, leading to branch migration in the direction of recA medidated strand exchange

Front 41

What is RuvC and what does it do?

Back 41

Endonuclease that resolves HJ by cutting two strands at consensus sequence

Front 42

What are transposons?

Back 42

DNA elements capable of moving around the genome. Found in almost all organisms

Front 43

How is transcription similar to replication? How is it different?

Back 43

Similarities: Free nucleotides needed, strand synthesized 5'-3', polymerase needed

Differences: NTPs used instead of dNTPs, no primer needed, RNA pol, only part of genome transcribed, only one strand transcribed, Uracil instead of Thymine

Front 44

What subunits make up the RNA pol core? What subunit do you add to make holoenzyme?

Back 44

alpha (2), Beta, Beta'.
Sigma completes the holoenzyme

Front 45

What are the four steps of transcription?

Back 45

Binding, Initiation, Elongation, Termination.

Front 46

How are new rNTPs added to the polymer chain?

Back 46

3' OH reacts with alpha phosphate of incoming rNTP, liberating pyrophosphate

Front 47

Through which groove does sigma contact the DNA?

Back 47

Major groove

Front 48

What two regions does sigma bind with in DNA?

Back 48

-10 box and -35 box

Front 49

What is the function of the UP element?

Back 49

strengthens binding of sigma to promoter, increasing specificity

Front 50

What occurs during transcription initiation?

Back 50

RNA pol unwinds DNA closed complex to form open complex (-10 to +3). Synthesis begins.

Front 51

what is the coactivator for CAP?

Back 51

cAMP

Front 52

what is the result of lacI not binding 100% to operon?

Back 52

leaky TXN, allowing the cycle to begin when glucose is gone and lactose is present

Front 53

What level of genetic regulation is most common in lower organisms?

Back 53

Gene organization (such as in operons)

Front 54

What are 5 levels of gene regulation?

Back 54

Gene organization, transcription, mRNA stability, translation, protein stability and modifications

Front 55

What genes provide negative and positive regulation in the lac operon?

Back 55

lacI (neg) and crp (pos)

Front 56

What two reactions does B-galactosidase catalyze in the lac operon pathway?

Back 56

1. Hydrolysis of lactose to glucose and galactose
2. Conversion of lactose to allolactose

Front 57

What are four types of regulation?

Back 57

repression, activation, attenuation, autoregulation

Front 58

what is a regulon?

Back 58

collection of genes not in the same operon that are co-regulated

Front 59

what occurs in the sos response regulon?

Back 59

DNA repair genes induced by DNA damaging agents

Front 60

what genes are controlled by the mal regulon?

Back 60

genes needed to metabolize maltose

Front 61

what are the two phases of bacillus growth?

Back 61

vegetative(growing + dividing) and mother(dormant stage, outer cell dies but endospore activated by favorable conditions)

Front 62

Describe endospore formation

Back 62

1.Occurs in certain species of soil bacteria.
2.Triggered by lack of nutrients.
3.Requires turning off of many vegetative genes and turning on of spore-specific genes.
4.Requires 3 sigma factors (s29, s30 and s32 or sE, sH and sC) in addition to the vegetative sigma factor (s43 or sA).

Front 63

the araC operon is regulated by:

Back 63

autoregulation (and DNA looping)

Front 64

what process is necessary to produce the sigma K gene?

Back 64

recombination

Front 65

what does sigma do?

Back 65

specifies binding to promoter

Front 66

what does SDS-polyacrylamide electrophoresis do that agarose doesn't?

Back 66

Denatures proteins and separates subunits

Front 67

what type of bacteria did mitochondria supposedly evolve from?

Back 67

alpha proteobacterium like organism

Front 68

Describe mitochondrial RNAP

Back 68

1. 1 subunit core, phage like enzyme
2. specificity factor for initiating TXN at promoter
3. Similar core enzyme in plant, animal, and fugal mitochondria, but different specificity factors
4. promoter is 9-10 bp
5. genes encoded in nucleus
4.

Front 69

What organelle has both bacterial-like and phage-like polymerase? (PEP and NEP)

Back 69

Chloroplasts

Front 70

Why is chloroplast TXN much more complex than mitchondrial TXN?

Back 70

Not only are chloroplasts larger and more complex organelles than mitochon., they also differentiate.

Front 71

What larger category of organelles do Chloroplasts fit into?

Back 71

Plastids

Front 72

What are the types of plastids? describe each

Back 72

1. Proplastids - precursor of all plastids, found in meristems
2. Etioplasts - form in shoots of dark green plants, distinctive internal structure
3. Chloroplasts - in all green tissues
4. Amyloplasts - prominent in roots, store starch, colorless
5. Chromoplasts - in mature fruit, lots of carotenoids, little chlorophyll

Front 73

Which RNAP is more important in proplastids? Why?

Back 73

NEP because in proplastids there is not much plastid gene expression.

Front 74

In which organelle is PEP important? Why?

Back 74

Chloroplasts, because PEP is more important for photosynthesis

Front 75

What are PEP and NEP?

Back 75

Plastid-encoded polymerase(bacterial-like) and nuclear-encloded polymerase(phage-like)

Front 76

How is PEP like bacterial polymerase?

Back 76

composed of a core with the same subunits and a sigma factor for specificity. rifampicin sensitive.

Front 77

What genes encode subunits of the PEP core?

Back 77

rpoA(alpha)
rpoB(B)
rpoC1 and rpoC2(B')

Front 78

what is the active compound in death cap mushroom? what does it bind to?

Back 78

RNAP II is most sensitive, then III, I not sensitive

Front 79

What do you gain with primer extension?

Back 79

high resolution analysis of the 5'end of an RNA transcript

Front 80

How does CAP-cAMP stimulate TXN?

Back 80

by promoting formation of closed complex

Front 81

What advantages may be conferred by CAP-cAMP bending DNA?

Back 81

1. Bending may promote interaction of RNAP with UP element far upstream
2. Bending may increase amount of DNA in contact with CAP-cAMP, giving greater binding and specificity

Front 82

Why does the lac operon need an activator?

Back 82

It does not have a strong core promoter

Front 83

compare and contrast mitochondrial and chloroplast RNAPs

Back 83

Mitochondrial RNAP: nuclear-encoded, one subunit and a specificity factor, phage-like

Chloroplast RNAPs: Contains both Plastid and Nuclear encoded RNAPs. The plastid encoded polymerase (PEP) is bacterial like, having the same subunits, sigma recognizing -10 -35 promoters, and rifampicin sensitivity as E. coli RNAP. The Nuclear encoded polymerase (NEP) is phage like, having one subunit , a specificity factor that recognizes a single 7-10 bp promoter, rifampicin insensitive.

Front 84

Where are monocistronic and polycistronic transcription found?

Back 84

monocistronic: eukaryotic nuclear
polycistronic: mammalian mtDNA
both: prokaryotes, chloroplast, mtDNA(except mammalian)

Front 85

From which type of bacteria did Chloroplasts arise?

Back 85

Cyanobacteria