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2013-04-30 00:59:17

dna binding
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  1. 1. What does nucleotide consist of? (3)
    2. What is a nucleoside?
    3. What is the link? What type of link is it? what is it between?
    4. What is the 3'OH end important for?
    5. Purines are small words and ____?
    1. Sugar (pentose), phosphate, and N base (purine or pyrimidine)

    2. Same as above w/o phosphate

    3. Phosphodiester: b/t alcohol (3' on sugar) and acid (phosphate)

    4. Replication and transcription

    5. Large structures
  2. 1. What is the difference b/t adenine & guanine?
    2. what's the difference between cytosine and thymine? Thymine and uracil?
    • 1. Guanine has a NH2 poking out of its left side.
    • 2. Thymine has CH3 poking out at 2 pm and has a C=O instead of NH2 at the top. Uracil doesn't have the CH3, but has the C=O

  3. 1. Why is DNA very stable?
    2. Half life of RNA?
    3. Where do nucleotides absorb?
    4. What are strands held together by? What connects strands?
    5. What is the approximate diamter of H-bonds?
    6. Why does A=T only have 2 H bonds?
    • 1. Lack of 2'OH group preventing hydrolysis.
    • 2. Minutes
    • 3. 260 nm
    • 4. Phosphodiester bonds. OH bonds.
    • 5. 3 A.
    • 6. B/c Adenine doesn't have an EN to make an H bond off of.
  4. 1. Which form of DNA is favored in dehydrated situations? What is different about this form from B?
    2. Which is the physiological form? Which is most stable?
    3. Which one is left handed?
    4. Looking at melting/denaturation curves of DNA specimens, where is the metling point?
    5. What does the melting point depend on? 4
    6. What increases melting point?
    1. A form - wider, more compact, base pairs are not perpendicular to central axis and more exaggerated grooves

    • 2. B, B
    • 3. Z
    • 4. The point at which 50% of the DNA is melted.
    • 5. pH, ionic strength, size, and base composition of DNA
    • 6. GC pairing
  5. 1. What are the 3 phases of DNA replication and transcription?
    2. How would you best describe replication? (3)
    3. Describe the Messelson Stahl experiment. What did it confirm?
    • 1. Initiation, elongation, termination
    • 2. Semi-conservative, high fidelity, and efficient
    • 3.

    Confirmed that DNA replication was semi-conservative
  6. 1. Where does DNA begin in bacteria? In eukaryotes?

    2. Is it bidirectional or unidirectional?

    3. In what direction is DNA read? In what direction is it synthesized?

    4. Where does replication occur? aka 3 other names

    5. What takes place simultaneously during replication?

    6. Define replication fork
    • 1. Single point of origin (in eukaryotes multiple points of origin)
    • 2. Can be either
    • 3. 3-->5. 5--->3
    • 4. Replication fork, eyes, bubbles, theta structures

    5. Unwinding and replication and rewinding

    6. Replication fork = replisome (complex of DNA and series of enzymes including DNAP) where replication & unwinding take place.
  7. 1. How does replication proceed?
    2. Why can elongation only occur in 5'-->3' direction?
    3. What direction is the lagging strand synthesized in relation to movement of replication fork? Leading strand?
    4. What is needed for DNA synthesis to begin? How many does lagging strand need? Leading?

    5. What is responsible for nicking out DNA ligases and filling them with a nucleotide?
    • 1. Semi-discontinously
    • 2. Because new nucleotides require Nu attack by 3'OH on existing strand and can only be added there.

    3. Same direction as replication fork. Opposite direction.

    4. DNA needs RNA primase to make a primer. one for each okazaki fragment. 1.

    5. DNA Pol I
  8. 1. Why is RNA primer needed?
    2. Why is Pol I not the main DNA replication enzyme?
    3. What is processivity? Who has the highest processivity? Who has the lowest?

    4. Which enzyme has 5'-->3' exonuclease activity? 3'-->5'?

    5. Who has the highest and lowest polymerization rate? Define polymerization rate.
    1. B/c DNA polymerases can only add nucleotides to pre-existing strand and RNA primer gives a strand segment, complementary to the template with a free 3'OH group for a nucleotide to be added

    2. Because its nucleotide addition rate is too slow

    3. Processivity = number of nucleotides added before polymerase dissociates. III = highest; I = lowest.

    4. DNA Pol I has 5'-->3' exonuclease activity which is unique to DNA polymerases. All the other ones have 3'-->5'

    5. Highest polymerization rate (nucleotides/s) is III. Lowest is I.
  9. 1. What ion is needed for DNA elongation?
    2. What do the ions do? (4)
    3. Define holoenzyme. What catalyzes synthesis of leading & lagging strand?
    4. What is the entire complex of enzymes & proteins required to synthesize DNA called?

    2. (1) Mg2+ on right facilitates attack of 3'OH of alpha phosphate to allow Nu attack. (2) Mg2+ on left stabilizes pyrophosphate promoting its displacement.  (3)Both ions stabilize structure of the pentavalent transition state and are (4) coordinated with 3 aspartate groups in DNAP.

    3. Holoenzyme - biochemically active compound formed by combo of an enzyme w/ coenzyme. DNA Pol III holoenzyme

    4. Replisome
  10. 1. What are the 2 main activities of DNA Pol III?
    2. Why is DNA Pol I processivity so slow?
    3. Is DNA Pol I enough to connect okazaki fragments?
    4. Why does bacterial cell require such a large complex for DNA replication?
    • 1. 5'-->3' elongation & proofreading activity
    • 2. B/c its job is to remove RNA primers and fill them back in.
    • 3. No, we need DNA ligase too to make the phosphodiester bond b/t nucleotide
    • 4. To ensure fidelity, b/c any imstakes in genetic replication leads to major problems for the organism.
  11. 1. What special enzymes/proteins are needed to ensure proper translation? 8

    2.Number of bp for E.Coli? Eukaryotes? Number of errors?

    3. How do non-pol I check? What is it highly specific for?

    4. Does proofreading require pyrophosphate?

    5. What requires ATP?
    1. DNA Pol III, DNAPI, primosome (DnaB protein (helicase)+ primase), DNA ligase, DNA gyrase (topoisomerase), ss binding proteins,

    2. 4.6 x 10^6, 3 billion. 1 in 1000 or 10,000

    3. Can only check the nucleotide that was just added on. Highly specific for mismatched base pairs.

    4. No

    5. Helicase
  12. 1. What are sources of fidelity? (3)

    2. Additional factors? (2)

    3. What is required for termination of replication in E.coli?

    4. Why do we need the above?
    1. dNTP concentrations (increased dNTP --> increased errors)

    2. 2-step polymerase reaction (binds correct dNTP and proofreading)

    3. Ter-Tus complex. There are Ter sequences that allow replication fork to enter, but cannot leave. Ter sequences function as binding sites for the Tus protein. Replication ends when replication fork comes across Ter in right order.

    4. Even though opposing replication forks generally halt when they collide, Ter-Tus may prevent overreplication by one fork in the event that the other is delayed or halted.
  13. 1. How do [dNTP] affect error rate?

    2. Does transcription use DNA-binding protien sand topoisomerases? Does it require a primer? Does it require helicase?

    3. What is the only macromolecule that not only stores/transmits info and catalyzes rxns?

    4. Are proteins important in transcription?

    5. What does DNA-dep RNA pol require? (5)
    • 1. Increased dNTP increases error rate
    • 2. Yes,no, no.

    3. Ribozymes

    4. Yes, all nucleic acids including RNAs are complexed w/ proteins

    5. DNA template, NTPs, precursors of the nucleotide units of RNA, Mg2+ and Zn2+
  14. 1. Are all genes transcribed at the same rate? What does this depend on?
    2. What are differences in transcription in eukaryotes vs. prokaryotes?
    1. No, depends on role of product protein in the cell

    2. Prokaryotes: no organized nucleus (distinct nucleus); dsDNA --> ssRNA --> protein in same place (transacription and translation are compartamentalized); eukaryotes have more processing of mRNA transcript
  15. 1. Are most RNAs m/t/rRNAs?
    2. What is the main role of rRNA? What % of the ribosomal mass is it? How often are they transcribed? By what type of genes?

    3. Less than 1/3 of all RNA is ____?

    4. What is the one enzyme required for prokaryotic transcription. What does it consist of? (4)
    1. No, most of them are not classified int hose categories.

    2. Integral structural component of ribosome (66%) the rest is other proteins. ALL THE TIME, by housekeeping genes.

    3. mRNA

    4. RNAP = large multisubunit enzyme (MW=~500,000) core enzyme (aBB'w) + sigma unit --> holoenzyme (aBB'ws), and Mg2+
  16. 1. What is non-template strand doing?
    2. How many bp are unwound at any time?
    3. What direction do RNAP and transcription bubble move in?
    4. How are NTPs funneled in?
    5. Write equation for this process - what is it similar to? What is special about this?
    • 1. It is being physically moved out of the way by something that starts with an h.
    • 2. 10-12 (17)
    • 3. 3'-->5' of the DNA
    • 4. From channel int he protein
    • 5.(RNA)n+ NTP <---> (RNA)n+1+ Ppi

    This is highly conserved in evolution.
  17. 1. How are genes organized in prokaryotes: How are protein-encoding genes arranged?

    2. Define operons

    3. How are the genes transcribed?

    4 How does RNAP know where to bind? What does it bind to? What is another word for this sequence? (2)
    1. Protein-encoding genes are arranged in tandem along a single strand and are transcribed as a single unit

    2. Gene "units" = operons

    3. The genes "cistrons" are transcribed as 1 long polycistronic mRNA

    4. Binds to sigma unit. Sigma unit is responsible for binding to -10 - Pribnow box and -35 (consensus sequence) of the promoter.
  18. 1. What is a strong promoter vs. weak promoter? How do variations in the consensus sequence affect transcription?

    2. How can a change in one base pair of the consensus sequence change rate of binding?

    3. What is gene expression controlled by in part?
  19. 1. Describe mechanism of DNA binding, initiation, elongation
    1. Polymerase, directed by sigma factor, binds to promoter leading to formation of closed complex (promoter DNA is stably bound to DNA, but not unwound)

    2. Positive end is unwound to form an open complex

    3. INITIATION: tx is initiated within complex, leading to conformational change that converts complex to elongation form. Transcription complex moves away from promoter

    4. Sigma subunit dissociates at random as polymerase enters dissociation phase and NUsA replaces it (competitive binding)

    5. Elongation is just like DNA's. Mg2+ coordinates to phosphate groups of incoming NTP and to 3 Asp residues on RNA Polymerase, which is highly conserved in all species. One Mg2+ facilitates attack by 3'OH group & the other facilitates displacement of the pyrophosphate & both stabilize pentacovalent transition state. Requires topoisomerases and ss binding proteins.
  20. Describe RNA termination

    1. Describe characteristics particular to rho indep termination

    2. What happens when DNA-RNA complex reaches the hairpin?
    1. Intrinsic (rho independent). Sequences intrinsic to DNA are expressed as RNA (1) DNA region that produces w/ self complementary sequences forming hairpin structure (2) highly conserved of 3 A residues in template strand that are transcribed into U residues.

    2. Disruption of interactions b/t RNA and RNAP --> dissociation.
  21. Describe extrinsic termination:

    1. Basic overview
    2. What is specific job of rho protein?
    3. Why is transcription complex stopped?
    4. How does rho protein translocate along RNA? What does rho require?
    5. How does rho protein promote release of RNA transcript?
    1. Protein (termination factor) binds to nascent RNA and causes physical dissociation of RNAP and DNA.

    2. Rho protein associates w/ RNA at specific binding sites and moves 5'-->3' until it reaches the transcription complex paused at termination site.

    3. RNA complex reaches CA-rich sequence called rut (rho utilization element) causing it to pause so rho protein can catch up to it.

    4. Rho protein has ATP-dep RNA-DNA helicase activity.  REQUIRES ATP!!!!
  22. 1. What comes off of the RNA-DNA complex when elongation ends?
    2. How many RNAP in eukaryotes? What does each one do? (1,3,3)

    3. What are other differences b/t consensus sequences in eukaryotic transcription vs. prokaryotic? 3
    1. Completed mRNA and NusA

    2. RNAI - makes preRNA RNAPII - mRNA, recognizes lots of promoters, makes specialized RNA; RNAPIII - tRNA, snRNA< 5srRNA

    3. No -10, -35 --> uses TATAbox instead (formation of closed comlex begins when TATA bidning protein binds to TATA box), GC box, and enhancers/silencers
  23. Intron splicing:
    1. What is needed in premRNA (3)
    2. What type of rxn does it need? How many? What else?
    3. What binds with branching point?
    • 1. 3' splice site, 5' splice site, branch site - will always have these splice sites - they are highly conserved.
    • 2. Nucleophilic attack (2); transesterification¬† to ligate the exons.
    • 3. 5' splice site
  24. 1. What does 5'cap require?
    2. What enzyme catalyzes this?
    3. What is the function of the 5'cap? (2)
    4. What is a splicesome? What is splicing the result of?
    5. Purpose of poly A tail? Enzyme? What does it require?
    • 1. GTP
    • 2. RNAPII
    • 3. To protect mRNA from ribonucleases and participates in binding of mRNA to ribosome to help initiate transcription
    • 4. Splicesome - complex of premRNA, snRNA, and proteins.

    5. Protection form enzymatic destruction. Polyadenylate polymerase. Requires ATP.
  25. 1. What is alternative splicing
    2. What does this allow?
    1. Different ways of expressing same genomic sequence in each cell. Some exons will be kept and others will be discarded

    2. Allows us to be economical with the amount of DNA we have to store