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CORE Block 2 > Transcription I > Flashcards

Transcription I Flashcards

1
Q

Where do enhancers lie with relation to the genes they affect?

A

Because of the flexibility of DNA, they do not need to be close in location or origin to the genes they regulate.

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2
Q

What percentage of the genome is actually transcribed?

A

About 6-8% of the genome is transcribed.

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3
Q

Why is RNA less stable than DNA?

A

Because we do not need the mRNA to stick around as long as DNA. RNA is more regulated.

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4
Q

Name two reasons that uracil is used in place of thymine in RNA.

A

It requires less energy to make than thymine, and thymine is easily replaced by mutations if cytosine were to be deaminated.

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5
Q

How is the process of transcription similar to that of DNA replication?

A
  • Need to open and unwind portion of the DNA helix.
  • One of the two strands of DNA acts as a template for synthesis of RNA molecule.
  • Nucleotide sequence of RNA chain is determined by complementary basepairing.
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6
Q

How much DNA is unwound at any given time for transcription to take place?

A

Only 17bp are unwound at a time (do not need to spend the energy to unwind the whole molecule)

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7
Q

How does the newly made RNA strand separate from the DNA strand?

A

As the DNA is rewound and the duplex reforms, the 8bp DNA-RNA hybrid is displaced and the RNA strand peels off.

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8
Q

How does RNA transcription differ from DNA replication?

A
  • RNA strand does not remain hydrogen bonded to DNA template (noncoding) strand. RNA strand is displaced and DNA helix reforms.
  • RNA molecules are much shorter than DNA molecules (few thousand versus hundred million)
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9
Q

How fast does RNA polymerase move?

A

50nt/sec

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10
Q

From where is the energy acquired to drive the addition of rNTPs to mRNA by RNA pol II?

A

From hydrolysis of high energy bonds.

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11
Q

What are some differences between RNA and DNA polymerases?

A
  • RNA uses rNTPs and DNA uses dNTPs
  • RNA polymerase does not need a primer to begin transcription
  • RNA polymerase has a lower fidelity than DNA polymerase (not necessary to get txn perfect, as it is transient)
  • RNA pol does not have proofreading mechanism
  • The polymerases are evolutionarily and structurally different
  • RNA polymerases are absolutely processive.
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12
Q

Describe the rules of complementary pairing in RNA transcription.

A
  • Uracil pairs with adenine.
  • methyl group of T is not involved in pairing with A
  • U occasionally pairs with G
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13
Q

What stabilizes the transition state of transcription?

A

Mg2+

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14
Q

What is the transcriptome?

A

It is the sum of all RNAs produced in a cell under a given set of conditions.

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15
Q

What percent of RNA is mRNA?

A

3-5%

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16
Q

Where does RNA pol II bind?

A

It binds in the promotor of the gene, but does not start transcribing here. The promotor also dictates the direction of transcription and the strand being transcribed.

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17
Q

What is the role of the mediator in RNA transcription?

A

The mediator binds together activator proteins on the enhancer with the RNA pol II.

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18
Q

What is the point of the lac operon in E. coli?

A

It allows bacteria to use different carbon sources other than glucose.

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19
Q

Under what conditions is the lac operon expressed?

A

Only when lactose is present in the cell and glucose is absent.

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20
Q

What is the role of the sigma subunit in RNA pol II?

A

It participates only in txn initiation and helps the RNA pol find the promotor, and makes sure that RNA polymerase binds stably only at the promotor.

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21
Q

Which DNA strand designates regulatory sequences for RNA txn?

A

The coding strand.

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22
Q

Describe the typical E. coli promotor.

A
  • TATA sequence at -10
  • TTGACA at -35
  • AT-rich element called UP and bound by RNA pol II alpha subunit
  • spacer regions vary in their number of nucleotides
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23
Q

Describe the process of initiation in prok txn.

A
  • Sigma factor of RNA pol II finds and bind the to the promotor sequence in duplex DNA. (closed complex)
  • RNA pol II unwinds duplex DNA transcription near start site forming a txn bubble. (open complex)
  • RNA pol II catalyzes addition of first 2 ribonucleotides
  • sigma factor falls off the promotor (promotor clearance)
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24
Q

Describe the events of txn elongation.

A
  • RNA pol II advances down the DNA, adding nucleotides in the 3’->5’ of the template strand.
  • at txn stop site, RNA pol II dissociates from the DNA and is recycled.
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25
Q

Describe intrinsic txn termination in prokaryotes.

A
  • upon reaching a terminator sequence, RNA pol II may pause and isomerize into a new conformation
  • nascent RNA transcript may form hairpin (GC rich) with itself that disengages the DNA-RNA hybrid and RNA pol II from DNA.
  • Complete RNA pol II dissociation from transcript occurs at AU hybrid region at 3’ end of new transcript
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26
Q

What does it mean for an mRNA transcript in prok to be polycistronic?

A

It means that 1 transcript contains more than one gene and more than one protein that are generally not post-translationally modified.

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27
Q

How is the large size of DNA in eukaryotes packaged?

A

Chromatin

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28
Q

How do transcription factors act on euk chromatin?

A

They can interact with chromatin to open it up to make it accessible to txn machinery including the large RNA pol II.

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29
Q

Which transcription factors impede txn by remodeling chromatin?

A

HDAC deacetylate histones so DNA remains bound. HMT methylates cytosines.

30
Q

Which txn factors allow for txn by remodelling chromatin?

A

HAT acetylates histones and allows for them to dissociate from DNA.

31
Q

Do core promotor regions in eukaryotes have universal consensus motifs?

A

No, they are highly diverse and complex.

32
Q

At what point in the euk promotor does DNA begin to be unwound?

A

At the initiator sequence (Inr)and TSS (transcription start site) is very closely downstream

33
Q

What are the initiation factors responsible for beginning initiation of txn in euk called as a group?

A

General transcription factors (GTFs). There are more than one of these, as opposed to the single sigma factor in prok txn initiation.

34
Q

How many GTFs are required for RNA pol II initiation?

A

Over 100 TFII’s.

35
Q

What is the general transcription apparatus in euk consist of?

A

GTF’s (TFII’s) and RNA pol II, and the apparatus interacts with the core promotor.

36
Q

What elements are found in the core promotor sequence in euk txn?

A

From 5’->3’:

  • TFIIB recognition element (BRE)
  • TATA box
  • Initiator (Inr)
  • motif ten element (MTE)
  • Downstream promotor element (DPE)
37
Q

Describe the CTD of euk RNA pol II

A

It has multiple repeats of YSPTSPS which is phosphorylated on serine 5 during txn.

38
Q

Describe the DNA-binding subunit of euk RNA pol II.

A

It does not bind sequence-specifically

39
Q

Describe the pre-initiation complex in euk txn.

A
  • TBP of TFIID binding TATA box
  • TFIIB binding BRE
  • TFIIF recruiting TFIIE and TFIIH to RNA pol II.
40
Q

Describe TFIID and its functions in euk txn.

A

It is saddle-shaped and TBP domain distorts DNA upon binding the minor groove (AT rich region allows for this as well). First step in txn initiaiton. Interacts with TFIIB and helps recruit the other transcription factors.

41
Q

How does TBP respond to upstream txn activators?

A

It cannot respond to them in any way.

42
Q

Describe TFIIB and its functions in euk txn.

A

It stabilizes TBP to the TATA box by binding to it, and also binds the BRE. Interacts with TFIIF also and helps recruit RNA pol II. It may be a target for regulatory txn factors.

43
Q

Describe TFIIF and its function in euk txn.

A

TFIIF along with TFIIB help recruit RNA pol II to the initiation complex. It attracts TFIIE and TFIIH to the complex. And it may promote elongation by remaining attached to RNA pol II during txn elongation.

44
Q

Describe TFIIE and its function in euk txn.

A

It recruits TFIIH to the initiation complex and modulates TFIIH kinase and helicase activities. It is required for escape of RNA pol II into elongation mode (promotor clearance)

45
Q

Describe TFIIH and its function in euk txn.

A

It is responsible for the open conformation of DNA + RNA pol II in initiation. TFIIH has ATP helicases which unwind DNA. Another TFIIH subunit phosphorylates serine 5 of CTD which assists in promotor clearance.

46
Q

Describe txn elongation and termination in eukaryotes.

A

Many txn factors such as TFIIE and TFIIH dissociate and elongation factors associate. After termination, RNA pol II dissociates from txn factors and its CTD is dephosphorylated by termination factors and is recycled.

47
Q

Is txn termination in eukaryotes DNA or RNA-directed?

A

It is mRNA directed.

48
Q

How are capping and splicing machinery recruited to the elongation complex in euk txn?

A

Phosphorylation and dephosphorylation of certain serine residues on RNA pol II CTD.

49
Q

How many protein-coding genes exist in the eukaryotic genome?

A

20-25,000.

50
Q

What causes different cell types to arise?

A

The different genes that they express.

51
Q

How can patterns of gene expression be used to identify cells of unknown origin?

A

Gene expression correlates with protein expression (not alwaus 1:1, however)

52
Q

At what different stages can protein expression be regulated?

A
  • transcriptional control
  • RNA processing control
  • RNA transport and localization control
  • translation control
  • mRNA degradation control
  • protein activity control
53
Q

Why does gene expression need t be controlled?

A
  • Developmental decisions
  • Maintenance of homeostasis (e.g. glucagon and insulin)
54
Q

What is one of the first steps in gene regulation?

A

Structural changes in a gene:

  • modification of DNA by methylation
  • modification of histones by methylation (TF’s can’t bind methylated histones) and acetylation
55
Q

Describe the role of specific transcription factors in txn regulation.

A

They selectively increase or decrease txn of specific genes, such as by binding to enhancers (can be far or close to txn start site (TSS).

56
Q

Describe combinatorial control.

A

Each gene has multiple enhancers. Combinatorial regulation by multiple elements and proteins is a central mechanism by which levels of gene expression are modulated.

57
Q

Describe cis-acting control elements.

A

They can be close or far away from the TSS. Can be in:

  • intergenic regions
  • enhancers
  • introns
  • close to TATA box or other initiator elements
58
Q

What are cis-acting elements responsible for?

A

Allowing the recruitment and assembly of the initiator complex.

59
Q

Describe the two critical domains of mammalian transcription factors.

A
  • DNA binding domain (DBD): recognizes and binds elements in a subset of genes
  • trans-activation domain (TAD): through a protein not encoded by the same gene. Recruits chromatid modifying enzymes to decondense the chromatin and allow general txn apparatus to interact with the start site. Mediates protein-protein interactions.
60
Q

Briefly describe txn regulation of the interleukin 4 (IL-4) in the immune response.

A

The IL-4 gene has a number of cis-regulatory elements that control the time and place when the gene will be transcribed.

61
Q

How do transcription factors recognize cis-regulatory sequences?

A

Euk TF’s have variety of structural motifs that interact with cis-regulatory sequences.

  • alpha helices lie in the major groove
  • sugar-phosphate backbone interactions
  • atoms in minor groove (some cases)
62
Q

Which proteins have some of the tightest interactions known in biology?

A

Transcription factors to cis-regulatory sequences.

63
Q

Which are the major families of TF’s?

A

They are categorized by the type of DNA binding domain:

  • Zinc finger
  • Helix-turn-helix
  • leucine zipper
  • Helix-loop-helix
64
Q

Describe zinc finger proteins and their role in txn.

A
  • Beta sheets and alpha helices held together by Zn
  • forms stretch of alpha helices along the major groove
  • multiple fingers may act cooperatively to bind nucleic acids (binding of other proteins, Zn helps with cooperativity)
65
Q

Describe the helix-turn-helix and its role in txn.

A
  • Txn factor DBD that is found in both prok and euk txn
  • Two alpha helices lying at 90 degree angle of one another, connected by turn
  • Recognition helix fits into major groove of DNA, other helices stabilize the interaction by making minor groove contacts
  • generally bind as a symmetric dimer, with each monomer having recognition and stabilizing helix
  • DNA binding site is two half-sites
66
Q

Describe the basic leucine zipper (b-ZIP) txn factor DBD and its role in txn.

A
  • DNA-binding dimer
  • formed from coiled-coil from two helices wound around one another (large hydrophobic residues, leucine)
  • extended alpha-helices grip the DNA like scissors in adjacent major grooves
  • many are heterodimers of alpha helices with different polypeptide chains
67
Q

Describe Helix-loop-helic DBD of TF’s.

A
  • large group of dimeric proteins containing basic region involved in DNA binding, and HLH region functioning as a dimerization domain
  • N-term alpha helices are basic and interact with DNA
  • C-termnal region has hydrophobic amino acids spaced at intervals to give amphipathic characteristic–basic HLH proteins
  • different HLH proteins can form heterodimers
68
Q

How does dimerization regulate function of HLH transcription factors?

A

-many tissue specific HLH proteins (class A) heterodimerize with ubiquitous HLH partners (class B) to become more stable and to bind DNA with higher affinity and to allow for variable cellular function.

69
Q

How do nonbasic HLH proteins affect DNA binding?

A

They prevent DNA binding.

70
Q

Why are bHLH transcription factors important in muscle development?

A
  • MyoD bHLH (but not non-basic HLH!) transforms fibroblasts into muscle.
  • MyoD kicks ID off E12 and E47 and initiates the muscle program
  • this family of genes shows that combinatorial associations of transcription factors can yield complexes with different functions
71
Q
A

CORE Block 2 (9 decks)

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