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Biology > Unit 4: Transcription And Processing Of RNA > Flashcards

Unit 4: Transcription And Processing Of RNA Flashcards

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1
Q
  • What is the central dogma of molecular biology?

4. 1

A
  • Processed associated with the flow of genetic information together with process of DNA replication
  • DNA=>RNA=>Protein
    • DNA to RNA = translation
    • RNA to Protein = transcription
    • DNA to DNA = replication
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2
Q
  • What are the role of main RNA related to transmission of gene information from DNA to proteins?
    4. 1
A
  • mRNA (messenger): transport information contained in genes to ribosomes
  • rRNA (ribosomal): most abundant, part of the structure of ribosomes
  • tRNA (transfer): transports aa for protein synthesis on ribosomes
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3
Q

What are some other RNA molecules/protein complexes involved in regulatory and catalytic functions?
4.1

A
  • snRNP (small nuclear ribonucleoproteins)

- micro-RNA

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4
Q
  • What are the function of the antisense DNA strand in transcription?
    4. 1
A
  • Different DNA strands have different functions in transcription
  • Antisense strand (3’-5’) used as a template for synthesise RNA molecule
  • so RNA sequence is identical to sense strand (5’-3’) with uracil instead of thymine and hydroxyl groups 2’C of pentoses
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5
Q
  • Why is cell differentiation associated with selective gene transcription?
    4. 1
A
  • Functions and conditions of a cell determine gene expression at any given time, so transcription is strictly controlled
  • Carried out through regulatory sequences, e.g *promoters, located at beginning of gene (5’ end of coding strand, 3’ end of template)
  • *Housekeeping genes: genes that are expressed in all cells of the body
  • *Inducible genes: only expressed in certain cell types and in variable ways. In some cases expression of gene is specific to 1 cell type
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6
Q
  • What is the role of RNA polymerase?

4. 1

A
  • MAin enzyme involved in transcription, responsible for RNA synthesis
  • Catalysed reaction: (NMP)n + NTP => (NMP)n+1 + PPi
    (NMP)n: RNA strand with “n” nucleotides monophosphate (NMP)
    NTP: nucleotide triphosphate
    PPi: pyrophosphate
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7
Q
  • What is the definition of Replication?

4. 1

A

A process that produces 2 identical strands of DNA from the original DNA molecule

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8
Q
  • How does initiation start?

4. 1

A
  • *Binding of RNA polymerase to promoter.
  • Promoter region extends 10s or 100s of bases before the *transcription initiation site to a few beyond point. There are *consensus sequences frequently repeated in different genes, even though promoters are highly varied.
  • DNA unwinds and polymerase undergoes conformational and chemical change due to *phosphorylation, inducing initiation of transcription.
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9
Q
  • How does elongation occur?

4. 1

A
  • Most transcription factors released at beginning of this phase.
  • RNA polymerase synthesised in 5’-3’ direction. 3’OH group of forming RNA reacts with phosphate of incoming ribonucleoside triophosphate, forming new phosphodiester bond.
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10
Q
  • How does termination occur?

4. 1

A
  • RNA synthesis ends when RNA polymerase recognises certain DNA sequences at ends of genes
  • Prokaryotes: Mechanisms extensively studied, sequences + factors participating in stopping of elongation and release of transcription machinery known
  • Eukaryotes: very few details of process known
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11
Q
  • Initiation in bacteria

4. 1

A
  • Bacterial RNA polymerase contains 5 subunits
  • *δ subunit identifies correct site for transcription initiation
  • Most bacteria have several diff. δ that direct polymerase to start sites in diff. conditions.
  • δ subunit relatively weakly bound so can dissociate from other subunits of core polymerase (See diagram pg. 14)
  • *Promoter: gene sequence that RNA polymerase binds to start transcription. (6 nt long, found 10-35 bp upstream of transcription start site)
  • *Consensus sequences: bases most frequently found in different promoters. (Mutations in these affect promoter function. Genes with promoters with differing sequences are transcribed less efficiently)
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12
Q
  • Elongation in bacteria

4. 1

A
  • After adding 10 nt, δ released from polymerase, leaves the promoter sequence and progresses on the DNA template to continue RNA elongation.
  • During elongation polymerase maintains unwound region of 15 bp
  • β + β’ subunits form crab-claw-like structure, gripping the DNA template. Channel between subunits contain polymerase active site of RNA synthesis
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13
Q
  • Termination in bacteria

4. 1

A
  • RNA synthesis continues till stop signal
  • *Most common stop signal: symmetrical inverted repeat of GC-rich sequence followed by 7 A residues
  • Results in *stable stem-loop structure in segment of RNA, disrupting association with DNA template => terminated transcription
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14
Q

When does most transcriptional regulation occur in bacteria?

4.2

A

Initiation

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15
Q
  • What is an operon?

4. 2

A
  • A unit made up of several linked genes which regulate other genes responsible for protein synthesis.
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16
Q
  • What is a Lac operon?

4. 2

A
  • *Enzymes involved in lactose metabolism
  • *Only expressed when lactose is present and no gluose. Enzymes involved:
    1. *lacZ (β-galactosidase): cleaves lactose into glucose and galactose
    2. *lacY (lactose permease): transports lactose into cell
    3. *lacA (transacetylase): function not clear
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17
Q
  • What is the negative control of the lac operon?

4. 2

A
  • Genes encoding these enzymes expressed as an *operon
  • 2 loci control transcription:
    - *i (outside operon): encodes represor protein that binds to operator
    - *o (operator): adjacent to transcription initiation site, where i gene product binds to operon
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18
Q
  • What is the central principle of gene regulation?

4. 2

A
  • *Control of transcription is mediated by the interaction of regulatory proteins with specific DNA sequences
  • Cis-acting control elements affect expression of linked genes on same DNA molecule (e.g. operator).
  • Other proteins can affect expression of genes on other chromosomes (e.g. the repressor)
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19
Q
  • What is negative control?

4. 2

A
  • *The regulatory protein (the repressor) blocks transcription
  • lac operon is an example of negative control
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20
Q
  • What is positive control?

4. 2

A
  • *Regulatory proteins activate transcription
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21
Q
  • What is the positive control of the lac operon?

4. 2

A
  • *Presence of glucose represses expression of the lac operon, even if lactose is present. So is mediated by positive control.
  • If glucose decreases, levels of cAMP increase. cAMP binds to regulatory *protein CAP (catabolite activator protein), an activator of lac operon.
  • CAP stimulated to bind to target DNA sequence upstream lac operon
  • *CAP facilitates binding of RNA polymerase to promoter. (RNA polymerase does not bind well alone so wont unless it has extra help from CAP)
  • *CAP only active when glucose levels are low (cAMP levels high). Thus *lac operon only be transcribed at high levels with no glucose
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22
Q
  • *What does the positive control of the lac operon ensure?

4. 2

A
  • **Bacteria only turn on the lac operon and start using lactose after they have used all their preferred energy source (glucose)

summary of lac operon use on pg. 23+24

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23
Q
  • What does RNA polymerase II synthesise?

4. 3

A
  • mRNA

- *Requires initiation factors that are not associated with the polymerase

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24
Q
  • What are general transcription factors?

4. 3

A
  • *Proteins involved in transcription of polymerase II promoters.
  • *Other transcription factors join DNA sequences that control the expression of individual genes

transcription factors = proteins involved in transcription that directly bind to DNA

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25
Q
  • What are the important sequence elements found in promoters?
    4. 3
A
  • *TATA box: resembles the 10 sequence of bacterial promoters. Consensus sequence=TATAA. 25-30 nucleotides upstream transcription start site
  • *Initiator element (lnr): Encompasses transcription start site (bit deformed so starts in middle)
  • *TFIIB recognition elements (BRE): about 35 nucleotides upstream transcription start site
  • *Downstream elements: DCE, MTE + DPE
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26
Q

What are the 5 transcription factors required for initiation of transcription in vitro?
4.3

A
  • *TFIID is composed of subunits, incl. *TATA-binding protein (TBP) and *other subunits (TAFs) that binds to the lnr, DCE, MTE + DPE sequences
  • *Other transcription factors *(TFIIB, TFIIF, TFIIE + TFIIH) bind in *association with RNA polymerase II to form the *transcription preinitiation complex
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27
Q

What are the steps required for initiation of transcription in vitro?
4.3

A
  1. Formation of transcription complex begins by binding of transcription factor TFIID. A subunit, the TATA-binding protein (TBP), binds to the TATA box; other subunits (TBP or TAF associated factors) bind to the lnr element and downstream promoter elements
  2. TFIIB binds to TBP + BRE elements
  3. Binding of TFIIF-associated polymerase
  4. TFIIB + TFIIH proteins associate with this complex. 2 TFIIH subunits are helicases, which unwind DNA around the initiation site. Another subunit is the protein kinase which phosphorylates serine residues in the C-terminal domain of the main subunit of polymerase II, inducing release of the polymerase and initiates transcription.
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28
Q
  • How is the preinitiation complex formed in vitro?

4. 3

A

See diagram on pg.36

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29
Q
  • What are the additional factors needed to initiate transcription in vivo?
    4. 3
A
  • RNA polymerase II/Mediator complexes + transcription initiation
  • Mediator complex is a protein made of 20+ subunits; interacts with general transcription factors + polymerase
    • stimulates basal transcription
    • key in association of general transcription factors with specific tf of certain genes that regulate gene expression
    • Released from polymerase on assembly of preinitiation complex and phosphorylation of C-terminal domain
    • Phosphorylated CTD then binds to other proteins that facilitate transcription elongation and are involved in mRNA processing
30
Q
  • How is the ribosomal RNA gene transcribed?

4. 3

A
  • *RNA polymerase I transcribes rRNA genes, in the tandem repeats
  • Transcription yields large 45S pre-rRNA which is processed to 28S, 18S +5.8S rRNAs
  • Promoters of rRNA recognised by 2 tf that recruit RNA polymerase I to form initiation complex: *UBF (upstream binding factor) + SL1 (selectivity factor 1)
31
Q
  • How are RNA polymerase III genes transcribed?

4. 3

A
  • genes for tRNAs, 5S rRNA + some snRNAs are transcribed by polymerase III. They are expressed from 3 types of promoters, which diff. Tf are bound:
    1. *5S rRNA gene promoter: *TFIIA initiates assembly of transcription complex by binding to specific sequences in promoter of 5S rRNA gene. *Then TFIIC, TFIIB + polymerase bind to the promoter.
    2. *tRNA gene promoter: *TFIIC bind and attracts TFIIB + polymerase.
    3. *Promoters of DNA encoding snRNA: SNAP + TFIIB bind cooperatively and polymerase is recruited.
32
Q
  • How is transcription regulated in eukaryotes?

4. 4

A

A- *The binding of proteins to specific regulatory sequences modulates transcription
- Sequences are identified by *gene transfer assays: Sequences are lighted to reporter genes that encode easily detected enzymes, e.g. firefly luciferase. Regulatory sequence directs expression of the reporter gene in cultured cells
B- Eukaryotic DNA packaged in chromatin which limits availability for transcription.
- *Non-coding RNAs + proteins regulate transcription via chromatin structure modifications

33
Q
  • What is the role of eukaryotic promoters?

4. 4

A
  • Genes transcribed by RNA polymerase II have 2 main promoter elements, which act as specific binding sites for general tf:
    1. TATA sequence (joined by TBP from TFIID)
    2. lnr (joined by TAF from TFIID)
  • Other cis-acting sequences serve as binding sites for variet regulation factors that control individual gene expression
34
Q
  • Other than promoters, how else are genes in eukaryotic cells controlled?
    4. 3
A
  • *Enhancers: regulatory sequences located farther from start site which tf bind to.
    0 ability of enhances to act from distance is due to *formation of loops in DNA
  • Activity does not depend on distance or orientation to transcription start site
  • First discovered in study of SV40 virus promoter: in addition to TATA sequences + set of 6 GC sequences, 2 repeats of 72 bp located upstream are requires to carry out efficient transcription

More info on pg. 44 if still confused

35
Q

*Give an example of where an enhancer is used

A
  • *Controls transcription of immunoglobulin (antibodies) genes in B lymphocytes.
36
Q

*What do enhancers represent in the human genomic DNA?

A
  • 10% or more of human genomic DNA.
  • There are many more enhancers than genes, which emphasise the importance of these elements.
  • Many human disease-related mutations affect enhancers rather than protein coding sequences.
37
Q

What does the immunoglobulin enhancer look like?

A

See pg. 46

38
Q

*How is the activity of an enhancer specific for the promoter of its target gene?

A
  • Specificity is maintained by the formation of discrete chromosomal domains: **topological association domains (TAD) (just remember name, not well studied yet)
39
Q

How do TADs work?

A

The frontiers of TADs contain *multiple binding sites for CTCF + FTIIC, which establish borders with cohesin

40
Q

What are transcription factors?

4.4

A
  • Proteins that bind to specific DNA sequences, thus controlling genetic information transcription
41
Q
  • What are the types of transcription factors?

4. 4

A
  1. *Basal (general) transcription factors: part of the preinitiation complex that interacts directly with RNA polymerase in the *promoter
  2. *Other transcription factors: differentials regulate gene expression by joining *enhancers. These are ensure genes are expressed in right cell at right time. They are *activators + repressors of transcription
42
Q
  • What do transcriptional activators do?

4. 4

A
  • Bind to regulatory DNA sequences and *stimulate transcription
43
Q

What are the independent domains of transcriptional activators?

A
  • 1 binds to DNA
  • Other stimulates transcription by 2 mechanisms
    1. *Interacts with Mediator proteins and general tf to facilitate assembly of transcription complex
    2. Interact with *coactivators to modify chromatin structure
44
Q

How do transcriptional repressors work?

A
  • Inhibit transcription
  • Compete with activators to bind to specific regulatory sequences
  • Inhibit through interactions with specific activator proteins, mediator proteins, general tf + corepressors; act by modifying chromatin structure
45
Q

How does chromatin affect transcription?

4.4

A
  • Limits availability of DNA DNA for transcription, affects both tf binding and action of RNA polymerase. Actively transcribed genes are in relatively decondensed chromatin.
  • Chromatin can be altered by histone modifications + nucleosome rearrangements
  • Man histone chemic modifications are stably inherited when cells divide, providing mechanism for transmission of gene expression patterns to daughter cells
46
Q
  • How can histones be modified?

4. 4

A
  • Histone acetylation: Amino-terminal end of histones extend outside of nucleosome. It is rich in lys and can undergo acetylation
  • Acetyl groups are added by *HAT (histone acetyltransferase) and removed by HDAT (histone deacetylase)
  • *Acetylatoin neutralises the positive charge of lys, *relaxing chromatin structure + increasing availability of DNA template strands for transcription
  • Transcriptional activators + repressors are associated with HAT + HDAT respectively
47
Q

In what ways can histones be modified?

4.4

A
  • Methylation of lys (K) + arg (R)

- phosphorylation of ser (S)

48
Q

How do histone modifications affect gene expression?

4.4

A
  • Alters chromatin properties

- Provides I ding sites for proteins that activate or repress transcription

49
Q
  • What are chromatin remodelling factors?

4. 4

A
  • Protein complexes that alter contact between DNA and histones
  • Can reposition, change the conformation of or eject nucleosomes from DNA
  • Can be incorporated into DNA in association with transcriptional activators or repressors, like histone modifying enzymes
50
Q
  • How do modified histones affect epigenetic inheritance?

4. 4

A
  • Modified histone serve as binding sites for proteins that catalyse histone modification (reason why pattern of modification can be inherited).
  • So histone modifications can regulate one another, leading to to the stable patterns of modified chromatin, providing mechanism for epigenetic inheritance (transmission of information not in DNA sequence)
  • Modified histones are transferred to progeny chromosomes to direct similar modification of new histones, maintaining characteristic patterns of modification

See diagrams pg. 57-58

51
Q

What is another method of epigenetic control of transcription?
4.4

A
  • Transcriptional repression
  • DNA methylation: addition of methyl groups at C5 of cytosine residues preceding guanines (CG dinucleotide)
  • Patterns are maintained after DNA replication
  • Methylation is clear and only represses but in histones it is unclear
52
Q

*What does DNA methylation play a role in?

A
  • Genomic imprinting
  • expression of some genes depend on whether they come from mother or father
  • e.g. Gene H19 is transcribed only from maternal copy. It is methylated during male development, but not female germ cells
53
Q
  • How do non coding RNA molecules regulate transcription?

4. 4

A
  • *miRNAs: (20-30nt) act by the RNA interference pathway to inhibit translation or induce degradation of homologous mRNAs
  • *lncRNAs (long nocoding RNAs): (>300nt) Form complexes with proteins that modify chromatin and recruit complexes to their sites of transcription, regulating expression of neighbouring genes (evidence not clear if can activate but can repress)
54
Q

How are bacterial mRNAs used?

4.5

A

-Immediately used for protein synthesis while still being transcribed

55
Q
  • Where are ribosomal RNAs derived from?

4. 5

A
  • Both prokaryotes and eukaryotes derived from single long pre-rRNA molecule
  • Prokaryotes: Cleaved to form 3 rRNAs (16S, 23S + 5S)
  • Eukaryotes: have 4 rRNAs; 5S transcribed from separate gene
56
Q
  • How do tRNAs begin?

4. 5

A
  • Start as long precursors (pre-tRNAs) for prokaryotes and eukaryotic
  • Cleavage of *5’ end of pre-tRNAs b enzyme *RNAse P
    • RNAse P is a ribozyme: RNA catalyses instead of protein
  • Cleavage of *3’ end by *RNAase protein
  • Addition of 3’ *CCA terminus, the site of aa attachment
  • Bases modified at specific positions. About *10& bases modified
57
Q
  • What happens to pre-mRNAs in eukaryotes before export?

4. 5

A

They are extensively modified

58
Q
  • How are eukaryotic pre-mRNAs modified before export?

4. 5

A
  • Throughout processing. Transport, translation + degradation, mRNA molecules are associated with proteins to form messenger ribonucleoprotein (RNA+protein=mRNA) particles
  • *Transcription and processing are coupled: CTD (C-terminal domain) of RNA polymerase serve as binding sitee for enzymes in mRNA processing
  1. Addition of *7-methylguanosine (modification) cap to *5’ end of transcript. 5’ cap stabilises RNA + aligns on ribosome in translation
  2. *poly-A tail added to 3’ end. Tail regulates mRNA translation + stability
  3. Introns are removed from pre-mRNA by *splicing
59
Q

What signals polyadenylation?

4.5

A
  • highly conserved hexanucleotide (AAUAA in mammals) located 10-30nt upstream polyadenylation site
  • GU-rich element downstream polyadenylation site
  • Some genes possess GU-rich sequence elements upstream hexanucleotide
60
Q

*What processing enzymes are associated with RNA polymerase CTD?

A
  • *Endonuclease cuts RNA strand at 10-30nt downstream of AAUAA, usually CA sequence(?)
  • *Poly-A polymerase then adds approx. 200nt of A
  • RNA synthesised downstream of poly-A addition site is degraded
61
Q

What are the 2 steps of splicing?

4.5

A
  1. Cleavage at 5’ splice site (SS) and joining of 5’ end of intron to an adenine within the intron (branch point). Intron forms a loop
  2. Cleavage at 3’ SS + simultaneous ligation of exons excises the intron loop
62
Q
  • Where does splicing take place?

4. 5

A

Large complexes called spliceosomes, which have 5 types of snRNAs - U1, U2, U3, U5 + U6

63
Q
  • How are snRNPs (small nuclear ribonucleoproteins) formed?

4. 5

A

snRNAs are complexed with proteins

64
Q

How are spliceosomes assembled?

4.5

A
  1. U1 snRNP binds to the 5’ SS. Recognition of 5’ SS involves bp between 5’ SS consensus sequence + complementary sequence at 5’ end of U1 snRNA
  2. U2 snRNP binds to the branch point
  3. Other snRNPs join complex and together form intron loop. While maintaining association of 5’ and 3’ exons so ligation occurs
  4. Excision of the intron
65
Q
  • What is the clinical importance of the correct recognition of splicing sequences?
    4. 5
A
  • estimated that 15% of genetic diseases are due to mutations affecting splicing sites
  • e.g β thalassemia: single nucleotide mutation in 1st intron of hemoglobin β chain Hebe generate defective protein, causing anemia
66
Q
  • Where do other splicing factors bind to?

4. 5

A
  • Bind to RNA and recruit U1 + U2 snRNPs to appropriate sites on pre-mRNA:
    • *SR splicing factors bind to specific sequences on exons + recruit U1 snRNP to 5’ SS
    • SR proteins also interact with *U2AF, which recruits U2 snRNP to the branch point
67
Q
  • What is alternative splicing?

4. 5

A

Most pre-mRNAs have multiple introns so *diff. mRNAs can be produced from same gene. One way of *controlling gene expression + *increases diversity of proteins that can be encoded

68
Q
  • What is RNA editing?

4. 5

A

Processing (other than splicing) that *alters protein coding sequences of mRNAs. Involves single base modification reactions, e.g deamination of cytosine to uridine and adenosine to inosine

69
Q

What is RNA turnover?

4.6

A

90% pre-mRNAs are intros which are degraded in nucleus after splicing. Processed mRNAs protected by capping a dn polyadenylation, but unprotected ends of introns recognised by enzymes and degraded

  • RNAs go to cytoplasm. Levels of RNA determined by balance between synthesis and degradation
  • rRNAs + tRNAs v. Stable in prokaryotes + eukaryotes. Accounts for high levels of these RNAs in cells
  • BActerial mRNAs degrade rapidly; most have 2-3 min half-lives. Rapid turnover allows quick response to changes in environment e.g. nutrient availability
  • Eukaryotic mRNA half-lives vary; <30mins - 2 hours. Short-lived mRNAs code for regulatory proteins, which rapidly respond to environmental stimuli. *mRNAs encoding structural proteins or central metabolic enzymes have long half-lives
70
Q

How are eukaryotic mRNAs degraded?

4.6

A
  • Degradation initiated by deadenylation (shortening the poly-A tails), followed by degradation from 3’ end or removal of 5’ cap and degraded from that end.

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