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Genomic Sequencing Technologies > Module 8.1 Transcription and RNA processing > Flashcards

Module 8.1 Transcription and RNA processing Flashcards

1
Q

RNA abundance by mass

A
  • rRNA (80-90%)
  • tRNA (10-15%)
  • mRNA (3-5%)
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2
Q

RNA by number of molecules

A
  1. tRNA
  2. rRNA
  3. other RNAs
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3
Q

Gene expression regulation

factors (5)

A
  • Which: gene function
  • Where: cell and tissue types
  • When: developmental stages
  • Quantity: expression level
  • Dynamic: external signals
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4
Q

Gene expression regulation

housekeeping genes

A
  • common to all cells
  • usually expressed universally in all the cells
  • genes coding for structural proteins of chromosomes
  • many proteins that form cytoskeleton eg. Actin
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5
Q

Gene expression regulation

Cell and tissue type

A
  • some RNAs and proteins only expressed in specialized cells
  • Hemoglobin expressed specifically in red blood cells where it carries oxygen.
  • tyrosine aminotransferase breaks down tyrosine in food expressed in liver but not in most other tissues
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6
Q

Gene expression regulation

Developmental stages

A
  • in early vertebrate development, Hox gene expressed at different times and locations
  • controls formation of body structures along anterior-posterior axis
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7
Q

Gene expression regulation

Expression level

A
  • level of RNA expression in different human cell lines of almost every gene found to vary from one cell type to another
  • genes expressed in all cell types usually vary in levels of expression in different cell types
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8
Q

Gene expression regulation

External signals

A
  • each cell is capable of altering pattern of gene expression in response to extracellular cues
  • starvation or intense exercise: Glucocorticoid released in body signals liver to increase production of sets of proteins involved in generating energy from amino acids and other small molecules
  • When hormone no longer present, protein production drops to normal unstimulated levels in liver cells
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9
Q

RNA polymerase

bacterial

A
  • multi subunit complex
  • subunit sigma factor is largely responsible for reading signals in promoter sequence that tells it where to begin transcription
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10
Q

RNA transcription

bacterial promoter

A
  • special sequence on DNA double helix that indicates starting position for RNA synthesis
  • heterogenous
  • two hexamers at -35 and -10 positions (relative to transcription start position) that are relatively conserved characteristic sequences recognized by sigma factor
  • nucleotide sequence between -35 and -10 hexamers differ among all promoter sequences
  • promoter sequence -> promoter strength
  • asymmetric - can only bind in one orientation
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11
Q

promoter strength

A
  • number of initiation events by promoter per unit of time measured
  • Promoters for genes of abundant proteins are much stronger than genes for rare proteins
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12
Q

Prokaryotic Transcription

process (7)

A
  1. RNA polymerase binds weakly to DNA and slides rapidly along DNA molecule until sigma factor recognizes promoter region
  2. polymerase binds tightly to promoter sequence
  3. Polymerase opens double helix to expose short stretch of nucleotides and use one of the two strands of DNA as template for RNA synthesis
  4. sigma factor dissociates and polymerase moves rapidly to elongate RNA chain until enzyme encounters terminator
  5. polymerase transcribes terminator sequence, hairpin structure forms and helps release RNA transcript
  6. polymerase dissociates from DNA
  7. polymerase reassociates with free sigma factor and searches for new promoter
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13
Q

Transcription regulator

A
  • regulates transcription initiation which respond to extracellular signals
  • Positions, identity, and arrangement of cis -regulatory elements determine time and place each gene is transcribed
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14
Q

transcriptional repressor protein

A
  • transcription regulator that turns genes off
  • genes that encode them continuously transcribed at low levels = fast response
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15
Q

transcriptional activator protein

prokaryotes

A
  • transcription regulator that turns genes on
  • poorly functioning promoters made fully functional by activator proteins that bind to nearby cis regulatory sequences and contact RNA polymerase to help it initiate transcription
  • binding of activator to DNA often controlled by introduction of a signaling molecule like a metabolite or other small molecule
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16
Q

Prokaryotic transcription

terminator signal

A
  • stretch of AT bases preceded by symmetric DNA sequence for most bacterial genes
  • when transcribed into RNA folds into hairpin structure
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17
Q

transcription activator proteins

eukaryotes

A
  • determines transcription rate and pattern
  • can sometimes act from several thousand nucleotides away from promoter region
  • help RNA polymerase, general transcription factors and mediators to assemble at promoter region
18
Q

eukaryotic transcription

5’ capping

process

A
  1. phosphatase removes one phosphate from 5’ end of new RNA
  2. guanyl transferase adds guanosine monophosphate in reverse linkage to 5’ end
  3. methyl transferase, adds a methyl group to guanosine
  4. Cap binds to a protein complex called CBP or Cap binding complex, which helps the RNA to be properly processed and exported.
19
Q

RNA splicing

A
  • Both intron and exon sequences are transcribed into RNA
  • The intron sequences are removed
  • The exon sequences are joined into a continuous coding sequence
  • machinery that catalyzes pre MRNA splicing involves multiple RNA molecules and hundreds of proteins.
  • complexity needed to ensure splicing is highly accurate yet sufficiently flexible to deal with enormous variety of introns
  • cell can easily regulate pattern of RNA splicing so that different forms of protein can be produced at different times in different tissues
20
Q

RNA splicing

process

A
  1. specific adenine nucleotide in intron region attacks 5’ splice site and cuts sugar phosphate backbone of RNA
  2. cut 5’ end of intron becomes covalently linked to adenine nucleotide -> loop in RNA molecule
  3. released free 3’ hydroxy end of Exon sequence then reacts with start of next exon sequence, joining exons
  4. intron is released and degraded
21
Q

Alternative splicing

A
  • Exons can be spliced in a variety of different ways to produce different mRNAs and proteins from the same gene
22
Q

mRNA 3’ end polyadenylation

process

A
  1. CSTF and CPSF proteins travel with RNA polymerase and bind to 3’ end processing sequence on RNA molecule as it emerges from RNA polymerase
    - CPSF =AAUAAA hexamer
    - CSTF = GU-rich region
    - third factor = 3’ CA sequence
  2. Additional proteins assemble for 3’ end modification
  3. RNA cleaved from cleavage site
  4. PolyA polymerase (PAP) adds ~200 A nucleotides one at a time to cleaved 3’ end
23
Q

Types of RNA

Messenger RNA (mRNA)

function

A

RNA that encodes proteins
transcribed from protein-coding genes

protein synthesis

24
Q

Types of RNA

Transfer RNA (tRNA)

A

RNA that functions as an adapter between mRNA and amino acids
selects amino acids and hold them in place on a ribosome

protein synthesis

25
Q

Types of RNA

Ribosomal RNA (rRNA)

A

RNA that forms the ribosome, the main machinery for protein synthesis

protein synthesis

26
Q

Types of RNA

Small nucleolar RNA (snoRNA)

function

A

RNA that facilitates chemical modification of RNAs

regulatory functions

27
Q

Types of RNA

MicroRNA (miRNA)

function

A
  • regulate gene expression by blocking translation of specific mRNAs and causing their degradation
  • > 1000 in humans
  • regulate >1/3 of all human protein coding genes
  • base pair with specific mRNAs and fine tune their translation and stability
  • guide sequences that bring destructive nuclease into contact with specific mRNA
  • single microRNA can regulate a whole set of different mRNAs, as long as mRNAs carry common short sequence complementary to microRNA

regulatory functions

28
Q

Types of RNA

Small interfering RNA (siRNA)

function

A

turn off gene expression by directing degradation of selective mRNAs and establishment of compact chromatin structures

regulatory functions

29
Q

Types of RNA

Long non-coding RNAs (lncRNA)

function

A
  • many serve as scaffolds
  • regulate diverse cell properties eg. X-chromosome inactivation

regulatory functions

30
Q

small nuclear RNA (snRNA)

A

Functions in various nuclear processes (eg. splicing)
directs splicing of pre-mRNA as part of maturation process

31
Q

Prokaryotic transcription

transcription regulators

A
  • protein that recognizes cis regulatory elements
  • binding event triggers reactions that specify which genes in cell are transcribed and at what rate
32
Q

Prokaryotic transcription

cis regulatory elements

A
  • specific sequence of DNA, typically 5-10 nucleotides
  • on same chromosomes as genes they control
  • usually dispersed throughout genome
  • Transcription of each gene controlled by its own complex arrangement
33
Q

Eukaryotic transcription

RNA polymerases

genes transcribed

A

RP I & III: tRNA, rRNA, various small RNA
RP II: most genes, including all those that encode proteins and many non-coding genes like micro RNA, siRNA, long non coding RNAs, and most small nuclear RNA genes

34
Q

Eukaryotic transcription

General transcription factors

features

A
  • needed at almost all promoters used by RNA Polymerase II
  • consist of set of proteins noted arbitrarily as TFIIA, TFIIB, TFIIC, TFIID, etc.
35
Q

Eukaryotic transcription

General transcription factors

assembly - purified DNA

A
  1. TFIID binds to TATA box with TATA binding protein (TBP) subunit
  2. TFIID binding causes large distortion in TATA box DNA
  3. Other factors and RNA polymerase II assemble to region to form complete transcription initiation complex.
  4. TFIIH with helicase subunit unwinds DNA to expose template strand.
  5. Phosphate groups added by TFIIH to tail of RNA polymerase (C terminal domain / CTD)
  6. Polymerase disengages from general transcription factor cluster
  7. RP II tightens interaction with DNA and acquires new proteins that allow long distance transcription
  8. Once elongation begins, most of general transcription factors are released from DNA
36
Q

TATA box

eukaryotes

A
  • short double strand DNA sequence primarily composed of T and A
  • typically located about 25 nucleotide upstream of transcription start site
37
Q

Transcription in eukaryotic cells

process

A
  1. transcriptional activators bind to specific sequences in DNA (enhancers that can be thousands of bp away) that help attract RP II to transcription start position
  2. mediator coordinates proteins at promoter region
  3. chromatin remodeling complex and histone modifying enzyme recruited to increase access to DNA in chromosome
38
Q

Eukaryotic transcription

mediator

A
  • large protein complex that coordinates assembly of all proteins at promoter region
  • allows activator to communicate properly with RP II and general transcription factors
39
Q

3’ poly A tail

CPSF & CSTF

A

proteins involved in mRNA 3’ end cleavage
- (CPSF) Cleavage and Polyadenylation Specificity Factor
- (CSTF) Cleavage Stimulation Factor

40
Q

Gene to protein overview

Eukaryotes vs Prokaryotes

A

Eukaryotes:
-mRNA transcribed by RNAP II
- 5’ capping, splicing, and polyA tail added in nucleus
- mature mRNA sent to cytoplasma for translation
- steps occur concurrently

Prokaryotes:
- 5’ and 3’ ends produced during transcription
- transcription and translation occur in common compartment
- translation often begins before mRNA synthesis has completed

41
Q

microRNA

formation/processing

A
  • 5’ capped and poly-A tailed
  • precursor forms double-stranded structure by base pairing with itself
  • cap and tail cropped off in nucleus and exported to cytosol
  • cleaved by dicer enzyme to remove loop -> 23 nucleotides double-stranded miRNA
42
Q

microRNA

mRNA processing

A
  1. miRNA associates with proteins to form RNA induced silencing complex (RISC)
  2. Argonaute protein and RISC components associate with both strands of microRNA, then cleaves and discards one strand
  3. argonaute holds 5’ end of miRNA and guides RISC complex to complementary mRNA 3’ UTR (>7 bases)
  4. If extensive base pairing: mRNA cleaved by argonaute protein to remove PolyA tail and expose to exonuclease
    - RISC and the associated microRNA released
  5. if less base pairing: inhibition of translation, mRNA destabilization, and eventually mRNA degradation

Genomic Sequencing Technologies (22 decks)

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