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Biochemistry III > Shirley's Notes Molecular Biochem I (Ben) > Flashcards

Shirley's Notes Molecular Biochem I (Ben) Flashcards

1
Q

What are the primary DNA polymerases involved in processive elongation of new DNA in prokaryotes?

And eukaryotes?

A

DNA polymerase III for prokaryotes

DNA polymerase ε for the leading strand and DNA pol δ for the lagging strand in eukaryotes

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2
Q

What are the proteins responsible for strand separation in the formation of replication bubbles…

in prokaryotes?

in eukaryotes?

A

prokaryotes - SSBs (single stranded DNA binding proteins)

eukaryotes - replication protein A (RPA)

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3
Q

What are the protein complexes responsible for unwinding of DNA in the formation of replication bubbles…

in prokaryotes?

in eukaryotes?

A

pro - a hexameric DNA β protein complex

euk - MCM complex

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4
Q

What do cyclins activate?

Which specific cyclins are associated with which of these cyclin-activated molecules?

What are the functions of these cyclin-activated thing complexes?

A

Cyclins activate cyclin dependent kinases (CDKs) which phosphorylate cell cycle-related molecules.

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5
Q

How are double-strand breaks in DNA repaired?

(What two methods? And what determines which method is used?)

A

Non-homologous End Joining is used during G0 or G1 phases.

Homologous recombination is used during S, G2, or M phases.

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6
Q

What important gene plays a role in “checkpoint control” DNA damage monitoring in G1/G2 phases of the cell cycle?

How?

A

Tumor Suppressor p53

  • normally very unstable, somehow stabilizes in presence of DNA damage
  • increased p53 activates transcription of cell-cycle delaying genes (including p21, a CDK-cyclin complex inhibitor that halts cell cycle progression)
  • if DNA damage is too extensive, cells undergo a p53-dependent apoptosis
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7
Q

What does DNA ligase bind to before performing strand ligation in prokaryotes?

In eukaryotes?

(Via what part of the enzyme and with the release of what?)

A

prokaryotes - binds NAD+ to make ligase-NMN

eukaryotes - binds ATP to make ligase-AMP

(P binds to enzyme via a lysine residue, releasing PPi)

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8
Q

Where does replication start in prokaryotes?

What characterizes this region?

A

Ori C

rich in A and T

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9
Q

What are the seven important components of the replicaton bubble?

A
  1. DNA A Protein - binds dsDNA to initiate unwinding
  2. SSB Proteins - bind ssDNA to prevent re-annealing
  3. Helicase (DNA B) - unwinds/separates strands w/ ATP
  4. DNA C Protein - w/ DNA B helps primase make primer
  5. Primase - makes 4-10 bp RNA primer
  6. Topoisomerase II - makes (-) supercoils w/ ATP
  7. DNA Polymerase III - main synthesis enzyme
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10
Q

How do topoisomerase I and II differ?

A

Type I cuts only a single strand of the DNA and reconfigures it to relax the double helix and is ATP independent

Type II cuts both strands to form negative supercoils in the dsDNA just ahead of the replication fork (which prevents its entanglement with the replicating ssDNA). It is ATP-dependent

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11
Q

What makes up the “primosome”?

A

DNA B (helicase) protein

DNA C protein

Primase

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12
Q

Describe the structure and function of polymerase alpha

A
  • Gap-filling
  • Lagging strand synthesis
  • has primase subunit (for RNA primers)
    • 4 subunits total
  • delta and epsilon take over elongation after alpha does a few 100 nucleotides

(analogous to polymerase I in prokaryotes)

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13
Q

Describe the functions of polymerases beta and gamma.

A

Beta - DNA repair (may have 3–>5 exonuclease)

Gamma - mitochondrial DNA synth + repair

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14
Q

Describe the functions of DNA polymerases delta and epsilon.

A

Delta - Lagginig strand (proofreads + repairs)

Epsilon - main leading strand synthesizer, can fill gaps and proofread, requires PCNA as its “sliding clamp”

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15
Q

Describe the function of telomerase.

What “template” sequence does it use to do its thing?

What sequence does it add (in vertebrates)?

A
  • adds a 5’-TTAGGG sequence to the 3’ free end of chromosomes after the removal of the RNA primer from the last Okazaki fragment (to prevent loss of coding DNA from the strand’s end)
  • contains a 3’-CAAUCCCAAUC-5’ RNA template
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16
Q

In what phase of the cell cycle does replication occur?

And how long does it take?

A

in S phase

about 8 hours

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17
Q

During replication, where do the histones of the original dsDNA molecule go?

Where are new histones added?

A

the “old” histones stay with the leading strand

the new ones are assembled into the lagging strand

(b/c histones only bind to dsDNA and the leading strand is more immediately dsDNA at the beginning of replication)

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18
Q

What is a nucleosome?

What connects nucleosomes?

What is a chromatosome?

A
  • Nucleosome = histone octamer (2 each of H2A + H2B + H3 + H4) plus ~140 nucleotides
    • 1.75 turns of DNA
  • linker DNA of ~60 nucleotides wrapped around H1 histone connects nucleosomes
  • Chromatosome = linker DNA + H1 + nucleosome
    • = 200 nucleotides + 9 histones
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19
Q

How many DNA molecules are there in a G2 phase cell?

A

92 molecules

  • double the usual 46 because DNA is fully replicated and mitosis is about to occur to split the nucleus in two
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20
Q

What are the six main types of DNA lesions?

And the general mechanism/factor causing each?

A
  1. Depurination - purine rings removed by heat/acid
  2. Deamination - amine removed from bases by radiation/alkylation (as in chemotherapy)
  3. Dimerization - same-strand adjacent Ts dimerize via UV radiation
  4. Deletion/Insertion - AKA frameshift, intercalating agents (histo dyes)
  5. Strand Breaks/Crosslinks - polymerase errors
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21
Q

What results from deamination of cytosine?

Of adenine?

A

Cytosine - becomes uracil + is complemented by adenine

Adenine - becomes hypoxanthine + comp’d by cytosine

22
Q

Why is thymine in DNA instead of uracil?

A

Because it’s easier for DNA repair mechanisms to distinguish between thymine and a de-aminated cytosine (which = uracil) when proofreading.

If uracil was supposed to be there, the proofreading would think de-aminated Cs were just normal Us.

23
Q

How is depurination repaired?

3 steps, 3 enzymes

A
  1. Endonucleases - cleave P-diester bonds @ missing purine
  2. Polymerase I / Beta - replaces missing purine base, but “nick” remains where P-diester is cleaved
  3. DNA Ligase - re-seals nick with new P-diester bond
24
Q

How is deamination repaired?

A
  1. DNA Gylcosylase - removes “odd” bases by breaking N-glycosyl bond btwn base and ribose
  2. Same 3 steps from depurination repair
    • endonuclease
    • polymerase I/B
    • ligase
25
Q

How is thymine dimerization repaired?

A

UV-specific endonucleases

  • activated by UV light, remove dimerized T-T
  • polymerase + ligase complete repair
26
Q

What is a mismatch and how is it repaired?

A
  • Polymerase mistakes can result in base mismatch and the resulting base pair can not form an H bond
  • In prokaryotes:
    • Parent strand recognized via its methylation
    • Repair enzyme MutS binds the mismatch and MutL finds the nearest methylation (MutH also helps somehow)
    • Helicase II + exonuclease unwind + digest daughter strand from methylation site to mismatch
    • DNA Polymerase III + Ligase complete repair
  • In eukaryotes:
    • Daughter strand recognized by nicks (parents strand not methylated)
27
Q

What are the two main types of point mutations?

And their sub-types?

A
  1. Substitutions
    • Transitions - base is subbed by same time of base
      • spontaneous, chemical or radiation causes
    • Transversions - pyrim subbed by purine or v.v.
      • polymerase error causes
  2. Frame Shift
    • ​InsertionorDeletion of base pair
28
Q

What are the three possibilities for results of a substitution mutation?

A
  1. Silent Mutation - mutated codon codes for same AA
  2. Missense Mutation - mutated codon codes for different AA
  3. Nonsense Mutation - mutated codon is stop codon
29
Q

What is the mechanism for spontaneous mutation?

Tell how this results in eventual substitution.

A
  • Normally oxo-form bases can spontaneously change to enol form which changes their complementary base
  • Example:
    • oxo-thymine changes to enol-thymine and its new complementary base is guanine
    • T-G base pair yields one normal T-A pair and one substitution mutation G-C pair upon second replication
30
Q

How is RNA transcription different in prokaryotes than eukaryotes?

7 items

A

​In prokaryotes…

  1. RNA transcript = mRNA without any modification
  2. mRNA is polycistronic
  3. TATA box promoter is closer to coding region
  4. mRNA has shorter life span
  5. transcription/translation happen in same place
  6. RNA polymerase can work alone (no transcr. factors)
  7. only one RNA polymerase exists (3 in euk.)
31
Q

What is the subunit make-up of E. coli RNA polymerase?

A

5 core subunits, 1 extra factor

  • 2 alpha subunits - recognize regulatory factors
  • beta subunit - has polymerase activity
  • beta prime - binds DNA
  • (omega subunit - unknown function)
  • sigma factor - non-core subunit, binds promoter region to allow transcription initiation
32
Q

Describe the initiation of transcription by E. coli RNA polymerase.

A
  1. polymerase binds DNA to seek promoter
  2. sigma factor binds promoter = close promoter complex
  3. polymerase unwinds DNA = open promoter complex
  4. first P-diester bond forms
  5. sigma factor dissociates + core subunits elongate RNA
33
Q

What are the two main consensus sequences in prokaryotic promoter regions?

A
  1. Pribnow Box (TATAAT) - 10 bases upstream
  2. TTGACA - 35 bases upstream
34
Q

Describe some structural features of polycistronic mRNA

A
  • pppA/pppG sequences before structural genes (5’ end)
  • AUG start codon (= formyl-Met)
  • start + stop codons btwn cistrons
  • 3’ end has non-coding region + terminator sequence
35
Q

What are the common eukaryotic consensus sequences for transcription promoter regions?

A
  1. TATA Box - (TATAAA @ -25 bp) binds obligatory transcription factors via TATA-binding protein
  2. CAAT Box - at -100 bp
  3. Gc Box - common on constitutive genes on template strand, at -45 bp, binds to SP1 transcr. factor
36
Q

Describe the eukaryotic RNA polymerases.

(location, products, inhibition)

A
  • RNA polymerase I - in the nucleolus​
    • makes rRNA (18s, 5.8s, 28s)
    • not inhibited by alpha-amantin
  • RNA polymerase II - in the nucleus
    • makes mRNA precursors + snRNA (for splicing)
    • strongly inhibited by alpha-amantin
  • RNA polymerase III - in the nucleus
    • makes rRNA (5s) and tRNA
    • weakly inhibited by alpha-amantin
37
Q

What are the 3 main primary transcript modifications in eukaryotic mRNA?

A
  1. 5’ Methylguanylate Cap - pppA/pppG loses a P + remaining PPi binds GMP which is methylated
  2. 3’ polyA tail - polyA addition signal shows endonuclease where to cut + then polyA polymerase adds ~250 As there using ATP (prolongs mRNA life)
  3. Splicing/Ligation - introns are spliced out + exons are connected
38
Q

Describe the 3 splicing sites for the removal of introns.

And which snRNA splices each one?

Briefly, how do snRNAs splice?

A
  • 5’ site - AG/GUAAGU (exon/intron)
    • U1-RNA splices
  • 3’ site - UUUUUUUUUXCAG/G
    • U5-RNA splices
  • Branch site - in middle of intron, varying sequence
    • U2-RNA splices
  • snRNAs transesterify by breaking one P-diester and forming another
39
Q

Describe the structure of tRNA and the function of its different parts.

A

Single stranded + partially double helixed with a secondary cloverleaf and tertiary L-shaped structure.

  • Acceptor arm - 5’ P + 3’ ACC to accept AAs
  • D loop/arm - binds aminoacyl-tRNA synthetase
    • has dihydrouridine
  • T loop/arm - binds ribosome
    • has TᴪC sequence (ᴪ = pseudouridine)
  • Anticodon loop/arm - binds codon
40
Q

How many codons are their for AAs?

And how many tRNAs?

How does this work?

A

61 codons

31 tRNAs

Because the tRNA anti-codon sequence often contains one non-binding modified base, so only two bases participate in codon-tRNA binding.

Most AAs have multiple codons with the same first two bases, so multiple codons can bind the same two-base tRNA anti-codon sequence.

41
Q

Describe the steps of “charging/activating” a tRNA with an amino acid.

A

Aminoacyl-tRNA synthetase has binding sites for AA, ATP and tRNA.

  1. AA and ATP bind synthetase.
  2. AA + ATP —> AA-AMP + PPi
  3. tRNA binds synthetase
  4. AA is transfered to 3’ end of tRNA, forming ester bond btwn AA -COOH and tRNA -OH groups
    • (either on C2 or C3 of tRNA’s 3’ ribose)
  5. AMP is released
42
Q

What are 2 important bacterial rRNAs and their general functions?

A
  1. 16s rRNA - part of the 30s small ribosome subunit, selects the protein synthesis start site (translation stops w/out it)
  2. 23s rRNA - part of 50s large subunit, interacts w/ tRNA to allow peptidyl transferase activity
43
Q

Describe the initiation of translation / ribosome assembly in prokaryotes.

A
  1. 16s rRNA binds a 3’ purine rich region before codons on mRNA
  2. Small 30s ribosome unit complexes w/ IF-1 + IF-3
  3. IF-2 binds GTP and complexes with fMET-tRNA
  4. All this complexes w/ 30s + IF-1/3 at its P-site
  5. IF-3 is released and 50s ribosome unit joins 30s
  6. GTP on IF-2 is hydrolyzed
  7. Remaining IFs are released and 50s + 30s assemble into 70s ribosome.
44
Q

What is the initiation codon for the first AA of every prokaryotic protein?

What is that AA?

A

AUG

codes for

formyl-Met

45
Q

What is the Shine-Dalgarno sequence?

A

the purine-rich (AGGAGG) sequence at the beginning of prokaryotic mRNA that attracts 30s binding for translation initiation

46
Q

Describe the initiation of translation in eukaryotes.

A
  1. eIF-2 binds GTP and tRNAf
  2. This complex binds 40s ribosomal unit
  3. 40s then binds 5’ methyl-guanylate cap on mRNA (with the help of eIF-3 and slides down to AUG start codon ( = Met )
  4. (Sometimes more 40s units line up btwn cap + codon)
  5. eIF-4 helps to unwind the mRNA at its 5’ end (using ATP)
47
Q

Briefly, what do the following initiation factors do…

IF-2

IF-3

eIF-2 through 4

A
  • IF-2 binds GTP and fMet-tRNA (prokaryotes)
  • IF-3 release from 30s allows 50s binding (prok.)
  • eIF-2 binds GTP and tRNAf (euk.)
  • eIF-3 helps 40s bind 5’ cap (euk.)
  • eIF-4 unwinds mRNA 5’ end using ATP
48
Q

What are the functions of the 3 prokaryotic and eukaryotic elongation factors?

A
  1. EF-Tu / EF-1α - brings aminoacyl-tRNA into A site using GTP
  2. EF-Ts / EF-1βγ - helps EF-Tu / EF-1α release GDP
  3. EF-G / EF-2 - is a translocase which uses GTP to move peptidyl-tRNA from A site back to P site
49
Q

What are the release factors in prokaryotes and their functions?

And in eukaryotes?

A
  • RF1 recognizes UAA/UAG stop codons
  • RF2 recognizes UAA/UGA stop codons
    • both 1 + 2 act at A site
  • RF3 stimulates binding of RF1/2 using GTP
    • RF binding alters peptidyl transferase such that it cuts peptide-tRNA bond to release PP chain, leading to 50s/30s dissociation
  • only eRF acts as RF in eukaryotes
50
Q

There are 6 basic possibilities for where a protein needs to go after translation, based on their signal peptides.

What are those 6 possibilities and what are the 2 possible intermediate destinations in which the protein will be placed before moving on?

A
  • Signal peptides sending proteins to nucleus, mitochondria or peroxisomes will cause proteins to first remain in the cytosol
  • Those sending proteins to the membrane, lysosomes or secretory vesicles will first send the protein to the RER
    • ​PTMs will occur in RER/Golgi before protein moves on

Biochemistry III (35 decks)

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