Download Transcription, RNA Processing, and

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Structure of RNA
Major Classes of RNA
Transcription in Prokaryotes
Transcription in Eukaryotes
Post-transcriptional Processing of Eukaryotic mRNA
Transcriptional Regulation in Prokaryotes:
the Lac Operon as an example
Transcriptional Regulation in Eukaryotes:
Steroid Hormones as an Example
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Uracil instead of Thymine
Ribose instead of Deoxyribose
Usually single-stranded
May have hairpin loops (e.g. loops in tRNA)
Messenger RNA
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mRNA
Contains information for the amino acid sequences
of proteins
Transfer RNA
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tRNA
Attaches to an amino acid molecule and interfaces
with mRNA during translation
Ribosomal RNA
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rRNA
Structural component of ribosomes
Small nuclear RNA
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snRNA
Component of small ribonucleoprotein particles
Processing of mRNA
Small nucleolar RNA
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snoRNA
Processing of rRNA
Small cytoplasmic RNAs
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Variable functions; many are unknown
Micro RNA
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miRNA
Inhibits translation of mRNA
Small interfering RNA
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siRNA
Triggers degradation of other RNA molecules
Piwi-interacting RNA
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piRNA
Thought to regulate gametogenesis
Requires a double-stranded DNA template
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The DNA strands separate, and only one of the strands is
used as a template for transcription
“Template strand” and “nontemplate strand”
Direction and numbering conventions
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From the 3’  5’ direction on the template strand is
called “downstream”
From the 5’  3’ direction on the template strand is
called “upstream”
The nucleotide at the transcriptional start site is
designated “+1” and the numbering continues +2, +3,
etc. in the downstream direction
The nucleotide immediately upstream from +1 is
designated “-1” (there is no 0); numbering continues -1, 2, etc. in the upstream direction
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Transcription requires nucleoside
triphosphates (NTPs; ATP, GTP, CTP, UTP) as
raw materials
Nascent RNA strand synthesis (elongation)
occurs only in the 5’  3’ direction, with new
nucleotides added to the 3’ end of the nascent
strand
Transcription is catalyzed by DNA-directed
RNA polymerases
The initiation of transcription occurs when RNA
polymerase binds to a “promoter region”
upstream from the transcriptional start site
Promoter regions typically have short stretches of
common nucleotide sequences, found in most
promoters, called “consensus sequences”
Common prokaryotic (bacterial) consensus
sequences include:
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-10 consensus sequence: TATAAT box or Pribnow box
-35 consensus sequence: TTGACA
-40 to -60: Upstream element; repetitive A-T pairs
Bacterial RNA polymerase consists of a core
enzyme and a sigma factor
Bacterial RNA polymerase core has 4 or 5
subunits
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α2ββ‘ω
α2ββ‘ is essential; ω is not
Sigma factors (σ) are global regulatory units.
Most bacteria possess several different sigma
factors, each of which mediate transcription
from several hundred genes …
… for example:
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In E. coli, during log (exponential) growth, the major
sigma factor present is σ70
During stationary phase, it is σS
Shifting from σ70 to σS activates the transcription of
multiple genes linked to survival during stationary
phase
Transcription begins when the core RNA
polymerase attaches to a sigma factor to form
a holoenzyme molecule
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The holoenzyme binds to a promoter, and the
dsDNA template begins to unwind
A nascent RNA strand is started at +1 on the
template
After transcription is initiated, the sigma factor
often dissociates from the holoenzyme
RNA polymerase moves 3’  5’ along the
template, synthesizing the nascent RNA
5’  3’
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Transcription ends (termination) when RNA
polymerase reaches a terminator sequence,
usually located several bases upstream from
where transcription actually stops
Some terminators require a termination factor
protein called the rho factor (); these are rhodependent. Others are rho-independent.
Messenger RNA in bacteria is often polycistronic,
which means that it has the code for >1 protein on
a single mRNA molecule; mRNA in eukaryotes is
almost always monocistronic
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Chromatin in eukaryotes is unfolded to permit
access to the template DNA during transcription
Eukaryotic promoters
Recognized by accessory proteins that recruit different
RNA polymerases (I, II, or III)
 Consist of a core promoter region and a regulatory
promoter region
 Core promoter region is immediately upstream from the
coding region
Usually contains:
TATA box – Consensus sequence at -25 to -30
and other core consensus sequences
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Regulatory promoter region
Immediately upstream from the core promoter, from
about -40 to -150
Consensus sequences include:
OCT box
GC box
CAAT box
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Eukaryotic RNA polymerases
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RNA polymerase I: Synthesizes pre-rRNA
RNA polymerase II: Synthesizes pre-mRNA
RNA polymerase III: Synthesizes tRNA, 5S rRNA,
and several small nuclear and cytosol RNAs
Also, the different RNA polymerases use different
mechanisms for termination
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In eukaryotes, mRNA is initially transcribed as
precursor mRNA (“pre-mRNA”). This is part
of a transcript called heterogeneous nuclear
RNA (hnRNA); the terms hnRNA and premRNA are sometimes used interchangably.
Almost all eukaryotic genes contain introns:
noncoding regions that must be removed from
the pre-mRNA. The coding regions are called
exons.
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Introns are removed, and the exons are spliced
together, by ribonucleoprotein particles called
spliceosomes.
mRNA contains a “leader sequence” at its 5’
end, before the coding region. The coding
region begins with a translational initiation
codon (AUG).
A methylated guanosine cap is added to the 5’
end of the mRNA by capping enzymes. The
cap is attached by a 5’  5’ triphosphate
linkage
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The coding region ends with one or more
translational termination codons (stop
codons).
At the 3’ end is a noncoding trailer region.
A 3’ poly-A tail, consisting of 50 – 250
adenosine nucleotides, is added to the 3’ end
by a 3’ terminal transferase enzyme.
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Operon: A group of genes in bacteria that are
transcribed and regulated from a single
promoter
Constitutive vs. regulated gene expression
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Constitutive gene expression: When a gene is always
transcribed
Regulated gene expression: When a gene is only
transcribed under certain conditions
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The lac operon in E. coli consists of:
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3 structural genes (genes that encode mRNA)
 lac z gene: Encodes β-galactosidase
 lac y gene: Encodes β-galactoside permease
 lac a gene: Encodes β-galactoside transacetylase
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The lac promoter gene: lac p
The lac repressor gene: lac i (constitutively expressed
and transcribed from its own promoter, different
from lac p)
The lac operator region: lac o (which overlaps lac p
and lac z)
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The genes of the lac operon are only
transcribed in the presence of lactose (or
another chemically similar inducer)
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In the absence of lactose, the lac repressor protein
binds to lac o (lac operator) and blocks RNA
polymerase from binding to the promoter (lac p)
In the presence of lactose:
 Lactose in the cell is converted to allolactose
 Allolactose binds to the lac repressor protein, causing it
to causing it to dissociate from the operator so RNA
polymerase can reach the promoter
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Transcription of the lac operon is stimulated
by conditions of low glucose concentration
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When glucose levels are low:
 Adenylate cyclase activity is high and the concentration
of cyclic AMP (cAMP) is high
 cAMP binds to the catabolite activator protein (CAP)
 The cAMP/CAP complex increases the efficiency of
binding of RNA polymerase to the promoter
 So there is increased lac transcription
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When glucose levels are high:
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Adenylate cyclase activity is lowered, so cAMP levels
are low
This means there is much less cAMP/CAP complex
And there is decreased lac transcription
So … E. coli will metabolize glucose first, then
lactose when the glucose runs out
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Steroid hormones are secreted by endocrine gland
cells and travel through the bloodstream
The steroid enters the cytoplasm of target cells
and binds to a cytoplasmic steroid receptor
protein
The steroid receptor/steroid complex enters the
nucleus, where it binds to regulatory sites
(typically upstream from specific promoters)
Transcription from some promoters may be
activated (“turned on”) while transcription from
other promoters may be inhibited (“turned off”)
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Once the genes that have been activated by the
steroid receptor/steroid complex (primary
response or early genes) have been transcribed
and translated, some of the proteins may act to
regulate the expression of other genes
(secondary response genes), etc.
So … you may have a series of different
transcriptional events over a time course with
early, middle, and late events