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Chemistry of Living Systems > Exam 4 (Ch 29-25) > Flashcards

Exam 4 (Ch 29-25) Flashcards

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1
Q

What is the Klenow fragment?

A

Region of DNA polymerase that contains a DNA binding cleft.

-Prokaryotic and eukaryotic RNA polymerase have similar fragments

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2
Q

What are the 5 functions of RNA polymerase?

A
  1. search for cis-acting elements
  2. Unwind a short segment of double helix
  3. Selects the correct ribonucleoside triphosphate (NTPs) and catalyzes phosphodiester bond formation
  4. Detects termination signals that say where transcription ends
  5. Interacts with activator/repressor proteins (i.e. transcription factors/trans-acting elements) that modulate rate of transcription initiation
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3
Q

What are cis-acting elements?

A

They are initiation/promoter sites that are on the same molecule as RNA polymerase

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4
Q

What are promoters?

A

Sequences of DNA that direct RNA polymerase to the proper initiation site for transcription/act as binding sites for transcription factors/accelerate transcription.

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5
Q

What/where are the 2 most common promoter sequences found on prokaryotic DNA?

A

Where: 5’ side of the start site
What: -10 and -35 sequence (centered 10 and 35 nucleotides upstream of start site)

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6
Q

What are the 3 stages of RNA synthesis?

A
  1. Initiation
  2. Elongation
  3. Termination
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7
Q

Describe footprinting and its function.

A

Function: to identify protein binding sites on DNA
How:
1. 32P labeled DNA is (un)treated with RNA polymerase and digested by DNase –> chain gets cut
2. Fragments separated via gel electrophoresis
3. Fragments made in absence of polymerase but not made in presence of polymerase show which bases were blocked by bound protein from hydrolysis

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8
Q

How does the sequence of the DNA coding strand compare to that of the RNA transcript?

A

They are the SAME with the exception of thymine (T) in place of uracil (U)

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9
Q

How does the sequence of the DNA template strand compare to that of the RNA transcript?

A

It is complementary to that of the RNA transcript

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10
Q

What are the other names that the coding strand and template strand known as?

A

Coding Strand= Sense (+) strand

Template Strand= Antisense (-) strand

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11
Q

Does RNA synthesis require a primer to start?

A

No, it can start de novo.

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12
Q

What is and where is the highly distinctive tag carried by newly synthesized RNA chains?

A

Where: 5’ end
What: First base at this end is either pppG or pppA
Significance: confirms that RNA synthesis starts at the 5’ end

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13
Q

What are the functions of each subunits of RNA synthetase holoenzyme?

A

Subunits (5): α2ββ’σ
σ=finds sites on DNA where RNA transcription can start
-breaks off from subunit once ~10 bases have been
assembled and can initiate further transcriptions
-dif types serve dif functions
(core enzyme) α2ββ’=used to complete transcription

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14
Q

Describe the elongation stage of RNA synthesis.

A
  1. NTP binds to active site of RNA polymerase
  2. NTP forms a Watson-Crick base pair with template strand
  3. Activates 3’-OH group of growing RNA chain
  4. Which then attacks the α-phosphoryl group
  5. Creates a phosphodiester bond and displaces PPi
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15
Q

What are the ways that RNA transcription can be terminated?

A
  1. Self Termination
  2. Termination Signals
    a. Hairpin structures
    b. Rho Factor
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16
Q

How can hairpin structures terminate RNA transcription?

A

Hairpin structure occurs where there are multiple uridines (U) in a row which is the weakest base pair interaction

-the RNA strand separates from the DNA strand and then from the polymerase thus ending synthesis

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17
Q

How does Rho factor terminate RNA transcription?

A

Rho factor=an ATPase enzyme

-it slowly pulls the transcript RNA (tRNA) away from RNA polymerase

18
Q

What are transcription factors?

A

Proteins required to initiate transcription

19
Q

What are enhancers/silencers?

A

Auxiliary nucleotide sequences in the template DNA (i.e. a cis acting element) to which activators and repressors bind

20
Q

What is the promoter for RNA Polymerase II?

A

TATA Box

21
Q

What are the functions of the following transcription factors: –TBP–TAFs–TFIIH–

A

TBP: a TATA binding protein that causes DNA to unwind and bend
TAFs: modulates rate of transcription
TFIIH: helicase that separates strands

22
Q

What is a preinitiation complex?

A

Preliminary structure of RNA polymerase consisting of a conglomerate of transcription factors that assemble around TBP

23
Q

Describe what occurs in RNA transcription initiation.

A
  1. Assembly of preinitiation complex (PIC)
  2. Phosphorylation of the carboxy terminal domain (CTD) to create an open PIC complex
  3. Once phosphorylated, CTD recruits RNA-processing enzymes
  4. RNA polymerase moves away from TATA initiation sequence and maj of transcription factors
  5. Left behind transcription factors can now assemble another PIC
24
Q

What are the 3 modifications that occur during posttranslational processing?

A
  1. Removal of polynucleotide segments by exo/endo-nucleases
  2. Addition of polynucleotide segments to the ends
  3. Modification of specific nucleotide residues
25
Q

Describe what occurs during mRNA processing.

A
  1. Caping via hydrolysis of 5’ triphosphate to diphosphate and attack at the α-phosphorous GTP to form a 5’-5’ triphosphate linkage
  2. Addition of polyA tail via poly(A) polymerase
  3. Editing via base change, deletions, insertions, and methylation of certain residues (to create diversity)
26
Q

What are introns and exons?

A

Introns: non-expressed intervening sequences that will be cut out

Exons: expressed sequences interspersed with introns which will be spliced together when introns are cut out

27
Q

What is characteristic of exon-intron junctions?

A

GU at the intron 5’ boundary and AG at its 3’ boundary

28
Q

What is the function of small nuclear RNAs (snRNPs)?

A

Found in spliceosomes, they catalyze the splicing of mRNA precursors

29
Q

What is the purpose of alternative splicing?

A

Purpose: to generate diversity
By splicing the same strand of pre-mRNA in different places, dif combos of spliced exons can be made which each will have a dif function

30
Q

What must be present for self-splicing to occur?

A

Self-splicing=spontaneous excision of an intron and splicing of 2 exons to form rRNA

Requires: GMP, GDP or GTP

31
Q

What mediates the 2 types of self-splicing reactions?

A

Group 1 Self-Splicing: mediated by a G cofactor

Group 2 Self-Splicing: mediated by 2’-OH group of a specific adenylate of the intron

32
Q

What are the functions of rRNA and proteins in ribosomes?

A

rRNA=makes up the active site

proteins=modulates catalytic activity and selectivity

33
Q

How many different triplet base code combinations are there?

A

4 nucleotide bases
Possible combinations= 4^3 = 64
ACTUAL COMBINATIONS= 4^2 = 16

34
Q

What is cell-free translation?

A

Method used to determine triplet code

  • cell contents are treated with DNase
  • mRNA allowed to spontaneously degrade
  • synthetic polynucleotide is added
35
Q

What are triplet binding assays used for?

A

Technique used to determine the coding specificity of a triplet of nucleotides

36
Q

What is the function of tRNA?

A

To act as an adapter molecule so that amino acids can interact/bind to nucleotides.

37
Q

What effect(s) does methylation have on translation?

A
  1. May prevent formation of certain base pairs –> making them accessible to other interactions
  2. Enhances the hydrophobicity of some regions which may be imp for interactions w/ synthetases or ribosomal proteins
38
Q

What is inosine? Where does it come from?

A

An unusual nucleoside found in anticodons; it maximizes the number of codons that can be ready by a particular tRNA molecule

-Made via: deamination of adenosine after the synthesis of the primary transcript

39
Q

Why is it important for an amino acid to be linked to a tRNA molecule prior to translation?

A
  1. Attachment of a given amino acid to a particular tRNA establishes the genetic code
  2. Formation of a peptide bond b/w free amino acids isn’t thermodynamically favorable so it must first be activated in order for protein synthesis to proceed
40
Q

What is the function of aminoacyl-tRNA synthetase?

A

To catalyze the activation of amino acids so that they can be linked to the correct tRNA molecule.
(functions= discriminate, activate, sometimes proofread)

Proofreading=to ensure that error rate is low
-does this via specific hydrolase activity which will hydrolyze any triplet codon that doesn’t match it

41
Q

what are the 2 classes of aminoacyl tRNA synthetase?

A

Class 1: acetylates 2’OH–>acyl migration to 3’OH (b/c approaches from the minor groove side)
Class 2: directly acetylates 3’OH

-Both interact w/ their tRNAs from the inside of the L and span the tRNA from the acceptor stem to the anticodon loop

Chemistry of Living Systems (7 decks)

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