Download Slide 1

RNA Classification
• Messenger RNA (mRNA) is produced by proteinencoding genes and is a short-lived intermediary
between DNA and protein
• It is the only type of RNA that undergoes translation
• Transcription of mRNA is often followed by posttranscriptional processing
1
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Functional RNAs
• Functional RNAs do not encode proteins, but
instead perform functional roles in the cell
• Transfer RNAs (tRNAs) are encoded in dozens of
forms and are responsible for binding an amino acid
and depositing it for inclusion into a growing protein
chain
• Ribosomal RNA (rRNA) combines with numerous
proteins to form ribosomes
2
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Additional Functional RNAs
• Small nuclear RNA (snRNA) of various types is
found in the nucleus of eukaryotes and plays a
role in mRNA processing
• Micro RNA (miRNA) is active in plant and
animal cells and is involved in postranscriptional
regulation of mRNA
3
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Additional Functional RNAs
• Small interfering RNA (siRNA) protects plant and
animal cells from production of viruses and
movement of transposons
• Certain RNAs in eukaryotic cells have catalytic
activity; these are called ribozymes
4
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
8.3 Eukaryotic Transcription Uses Multiple
RNA Polymerases
• Eukaryotes have three different RNA polymerases
that recognize different promoters and produce
different types of RNAs
5
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Eukaryotic Transcription
• Eukaryotic genes carry introns and exons, and
require processing to remove introns
• Eukaryote DNA is associated with proteins to form
chromatin; the chromatin composition of a gene
affects its transcription
• Chromatin thus plays an important role in gene
regulation of eukaryotes
6
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Eukaryotic Polymerases
• RNA polymerase I (RNA pol I) transcribes three
ribosomal RNA genes
• RNA polymerase II (RNA pol II) transcribes protein
coding genes and most small nuclear RNA genes
• RNA polymerase III (RNA pol III) transcribes tRNA,
one small nuclear RNA, and one ribosomal RNA
7
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Eukaryotic Polymerases, continued
• Each eukaryotic (and archaeal) RNA polymerase
contains units that share homology with the 5
subunits of the bacterial polymerase
• Arachaea and eukaryotes have 6 to 11 additional
subunits
• All RNA polymerases share a similar “hand” shape
with “fingers” that grasp DNA and a “palm” where
RNA synthesis takes place
8
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Cis-Acting Regulatory Sequences Bind
Trans-Acting Regulatory Proteins to Control
Eukaryotic Transcription
• Activator proteins bind regulatory sequences to
stimulate transcription
• Repressor proteins bind other sequences to hinder
transcription
• The regulatory proteins are found in large
complexes in eukaryotes
9
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Transcriptional Regulatory Interactions
• Three sets of regulatory DNA sequences are
commonly involved in eukaryotic gene regulation
• The core promoter region, containing the TATA box
and other sequences, is immediately adjacent to the
start of transcription; these bind RNA polymerase II
and its associated transcription factors
• Upstream of the core promoter region are various
proximal elements that bind regulatory proteins
• The last group of regulatory sequences are
“enhancers”
10
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Cis-Acting Regulatory Sequences and
Trans-Acting Proteins
• All three regulatory regions contain cis-acting
regulatory sequences, which regulate transcription
of genes on the same chromosome as the
sequences
• RNA polymerase II (Pol II) and various general
transcription factors (GTFs) bind the core promoter;
these are trans-acting regulatory proteins, which
can bind to their target sequences on any
chromosome
• At enhancers, aggregations of multiple proteins form
large complexes called enhanceosomes
11
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
12
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Promoter Elements
• The most common eukaryotic promoter consensus
sequence is the TATA box, or the GoldbergHogness box, located at about position 25
• The consensus sequence is 5-TATAAA-3
• A CAAT box is often found near the -80 position
• A GC-rich box (consensus 5-GGGCGG-3) is
located at 90, or further upstream
13
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
14
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Eukaryotic Promoter Elements
• Eukaryotic promoters display a high degree of
variability in type, number, and location of consensus
sequence elements
• The TATA box is most common, whereas the CAAT
box and GC-rich box are more variable
15
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
16
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Promoter Recognition
• RNA pol II recognizes and binds to promoter
sequences with the aid of proteins called
transcription factors (TFs)
• TFs bind to regulatory sequences and interact
directly, or indirectly, with RNA polymerase; those
interacting with pol II are called TFII factors
• The TATA box is the principle binding site during
promoter recognition
17
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Promoter Recognition, continued
• At the TATA box, TFIID, a multisubunit protein binds
the TATA box sequence
• The assembled TFIID bound to the TATA box forms
the initial committed complex
• Next, TFIIB, TFIIF, and RNA pol II join the complex
to form the minimal initiation complex
18
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Promoter Recognition, continued
• The minimal initiation complex is joined by TFIIE and
TFIIH to form the complete initiation complex
• The complete initiation complex contains multiple
proteins commonly referred to as “general
transcription factors”
• The complete complex directs RNA pol II to the 1
position, where it begins to assemble mRNA
19
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
20
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Enhancers and Silencers
• Promoters alone may not be sufficient to initiate
eukaryotic transcription
• Two categories of DNA regulatory sequences lead
to differential expression of genes
• These are enhancer sequences and silencer
sequences
21
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Enhancer Sequences
• Enhancer sequences increase the level of
transcription of specific genes
• They bind proteins that interact with the proteins that
are bound to gene promoters, and together the
promoters and enhancers drive gene expression
• Enhancers may be variable distances from the
genes they affect and may be upstream or
downstream of the gene
22
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Enhancer Sequences and DNA Bending
• Enhancer sequences bind activator proteins and
associated coactivators that form a “protein bridge”
that links the proteins at the enhancer sequence to
the initiation complex at the promoter
• This bridge bends the DNA so that the proteins at
both locations are brought close enough together for
them to interact
• This bridge is known as an “enhanceosome”
23
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
24
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Silencer Sequences
• Silencer sequences are DNA elements that act at a
distance to repress transcription of their target genes
• Silencers bind transcription factors called repressor
proteins that induce bends in DNA
• These bends reduce transcription of the target gene
• Silencers may be located variable distances from
their target genes, either upstream or downstream
25
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Insulator Sequences
• Insulator sequences are cis-acting sequences
located between enhancers and the promoters of
genes that need to be protected from the action of
the enhancers
• Insulators ensure that only the target gene is
regulated by the enhancer
• Insulators may allow formation of DNA loops that
contain the enhancers and their intended target
promoters while excluding nontarget genes
Genetics Analysis: An Integrated Approach
26
Copyright © 2012 Pearson Education Inc.
27
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Transcription Factors and Signal
Transduction
• Tissue-specific and developmental gene expression
are also affected by the presence of specific
transcription factors and signal transduction
pathways
• These pathways communicate the need for the
specific regulatory molecules, such as the specific
transcription factors needed for a particular gene
• Distinct transcription factor proteins are pivotal in
regulating transcription of such genes
28
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Transcription Factor Synthesis
• Transcription factor availability is dependent on their
transcription and translation in the cell
• Synthesis of transcription factors is tightly regulated
and different cell types have distinct arrays of
transcription factors
• The availability of activated transcription factors is
also controlled through signal transduction
pathways
29
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Signal Transduction
• Signal transduction pathways are sequential events
that release regulatory molecules inside a cell in
response to events outside the cell
• They utilize transmembrane proteins, which receive
signals externally through an extracellular interaction
domain
• They transmit signals within the cell via a binding
domain inside the cell; this activates a transcription
factor needed for expression of a target gene
30
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
31
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Enhancer-Sequence Conservation
• Implies selection to retain function
• Enhancers for particular proteins, such as binterferon, are conserved among mammals
32
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
33
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Yeast Upstream Activator Sequences
• In the yeast Saccharomyces cerevisiae, transcription
of genes in the galactose utilization pathway are
carefully regulated by enhancer-like sequences
• When galactose is the only sugar available, wild-type
yeast induce transcription of four enzyme-producing
genes, GAL1, GAL2, GAL7, and GAL10
• Together these import and then break down
galactose
34
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
35
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Upstream Activator Sequences
• Each of the GAL genes has its own promoter
and similar enhancer-like sequences that are
bound by a regulatory protein, Gal4, encoded by
the GAL4 gene
• The enhancer-like element is called the upstream
activator sequence (UAS, or UASG)
• Gal4 is continuously present in cells and interacts
with the Gal80 protein that binds Gal4 and keeps it
inactive in the absence of galactose
36
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Gal4 Function In the Absence of Galactose
• Each UASG element contains two 17-bp repeat
sequences that are binding sites for Gal4
• Gal4 functions as a homodimer with each
polypeptide forming two active domains
• One binds the 17-bp target sequence and the other
interacts with Gal80
• When Gal4 is bound to Gal80, the Gal4 DNA-binding
domain is unable to bind UASG
37
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
38
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Gal4 Function in the Presence of Galactose
• When galactose is present, galactose and Gal3,
encoded by the GAL3 gene, bind to Gal80
• Gal80 releases Gal4, freeing the DNA-binding
domain of Gal4 to recognize and bind to the UASG
sites
• The transcriptional activation domain then activates
transcription of the GAL genes
39
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
40
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Gal4 Activation of Transcription
• Gal4 binding of UASG leads to formation of a
multiprotein complex called the Mediator
• The Mediator is an enhanceosome that induces
formation of a DNA loop, making contact with the
general transcription apparatus at the GAL gene
promoters
41
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Repressor Proteins and Silencer Sequences
• Eukaryotic repressors inhibit transcription through
different mechanisms than those seen in bacteria
• One mechanism is the binding of repressor proteins
to silencer sequences, cis-acting regulatory
sequences that directly prevent enhancer-mediated
transcription
• The GAL genes utilize such a mechanism; the
protein Mig1 is produced in the presence of glucose
and binds silencer sequences upstream of the GAL
genes
42
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Repression of the GAL Genes
• The silencer sequences of the GAL genes are
located between the UASG sequences and the
promoters
• When bound, Mig1 attracts the protein Tup1 and the
two together form a repressor complex that prevents
UASG from initiating transcription
43
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
44
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Locus Control Regions
• The human b-globin gene encodes the b-globin
polypeptide, two copies of which join with two copies
of a-globin to form hemoglobin
• It is one of six closely related globin genes forming
the b-globin complex, with a locus control region
close to it
• A locus control region (LCR) is a highly specialized
enhancer that regulates transcription of multiple
genes packaged into complexes of closely related
genes
45
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
46
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
The b-Globin Locus Control Region
• The LCR regulating the b-globin complex contains
four distinct sequences, named HS1 to HS4
• These work together to produce the correct
expression of each type of b-globin gene throughout
development
• Each gene of the complex produces a distinct
polypeptide with unique oxygen-carrying capacity
47
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
The b-Globin Locus Control Region,
continued
• The HS1 to HS4 components bind regulatory
proteins that direct the formation of small DNA loops
• These serve as a bridge to the promoters of the
b-globin complex genes
• The composition of enhanceosomes bound to the
LCR at different developmental stages is varied,
such that the appropriate genes are expressed at
each stage
48
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
49
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
50
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Enhancer Mutations
• Mutation in the a- and b-globin genes produce
hereditary anemias called thalassemia
• Some thalassemia patients were identified with no
discernible mutations in the protein-coding part of
the globin genes, nor in their promoters
• It was found that some thalassemia cases were due
to deletions that altered the LCR regions, causing
abnormal expression of the globin genes
51
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Transcriptional Regulation by Enhancers
and Silencers
• Occasionally the same sequence can act as an
enhancer or a silencer, depending on which
regulatory proteins are present and bind to the
sequence
• Sometimes the enhancer or silencer sequence is
very distant from the gene it regulates
• A model for understanding transcriptional regulation
must include a mode of action of enhancers and
silencers and accommodate the variable distance
and position they occupy relative to the target gene
52
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
A Model for Control of Eukaryotic
Transcription
• The Sonic hedgehog (SHH) gene in humans and
mammals directs limb formation under an enhancer
1 million base pairs away from the SHH gene
• SHH is expressed in tissue-specific fashion due to
the action of two different enhancers
• One combination of regulatory proteins binds the
brain enhancer in brain tissue, but a different
combination binds the limb enhancer in developing
limbs
53
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
54
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
RNA Polymerase I Promoters
• RNA polymerase I transcribes genes for rRNA using
a mechanism similar to that of RNA pol II
• RNA pol I is recruited to upsteam promoter elements
following binding of transcription factors, and
transcribes ribosomal genes found in the nucleolus
• The nucleolus is a nuclear organelle containing
rRNA and multiple copies of genes encoding rRNA
55
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
RNA Polymerase I Promoters, continued
• Promoters recognized by RNA pol I have two
functional sequences near the start of transcription
• The core element stretches from 45 to 20, and is
needed for initiation of transcription; it is bound by
sigma-like factor 1 (SL1) protein
• The upstream control element spans from 100 to
150, and increases the level of transcription; it is
bound by upstream binding factor 1 (UBF1)
56
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
57
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
RNA Polymerase III Promoters
• RNA polymerase III is primarily responsible for
transcription of tRNA genes, but also transcribes one
rRNA and other RNA-encoding genes
• Small nuclear RNA genes have three upstream
elements whereas the 5S rRNA and tRNA genes
each have two internal promoter elements
• These are downstream of the start of transcription
58
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
RNA Polymerase III Promoters—The
Upstream Elements
• The upstream elements of the snRNA genes are a
TATA box, a promoter-specific element (PSE), and
an octamer (OCT)
• A small number of transcription factors bind these
elements and recruit RNA pol III
• RNA pol III initiates transcription in a manner similar
to other polymerases
59
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
60
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
RNA Polymerase III Promoters—The
Internal Promoter Elements
• Genes for 5S rRNA and tRNAs have internal
promoter elements called internal control regions
(ICRs)
• These are two short DNA sequences, called either
box A and box B for some genes, or box A and
box C for other genes
• These are located between positions 55 and 80
61
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
62
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
ANIMATION: mRNA Production in Eukaryotes
63
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Post-Transcriptional Processing
•
The initial eukaryotic gene mRNA is called the premRNA whereas the fully processed mRNA is called
the mature mRNA; modifications include
1. 5 capping
2. 3 polyadenylation
3. Intron splicing
64
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Capping 5 mRNA
• After the first 20 to 30 nucleotides of mRNA have
been synthesized, a special enzyme, guanylyl
transferase, adds a guanine to the 5 end of the
pre-mRNA
• Additional enzyme action methylates the newly
added guanine and may also methylate nearby
nucleotides of the transcript
• The addition of the guanine to the mRNA is called
5 capping
Genetics Analysis: An Integrated Approach
65
Copyright © 2012 Pearson Education Inc.
Functions of the 5 Cap
1. Protection of mRNA from rapid degradation
2. Facilitating transport of mRNA out of the nucleus
3. Facilitating subsequent intron splicing
4. Enhancing translation efficiency by orienting the
ribosome on the mRNA
66
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Polyadenylation of 3 Pre-mRNA
• Termination of transcription by RNA pol II is not fully
understood
• The 3 end of the pre-mRNA is created by enzyme
action that removes a section of the 3 message and
replaces it with a string of adenines
• This is thought to be associated with the subsequent
termination of transcription
67
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Steps of Polyadenylation
1. Cleavage and polyadenylation specificity factor
(CPSF) binds near the polyadenylation signal
sequence—5-AAUAAA-3—which is downstream
of the stop codon
• This is quickly followed by binding of cleavagestimulating factor (CstF) to a uracil-rich region
downstream of the polyadenylation signal sequence
• Two other cleavage factors, CFI and CFII, and
polyadenylate polymerase (PAP) also bind
68
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Steps of Polyadenylation, continued
2. The pre-mRNA is cleaved 15 to 30 nucleotides
downstream of the polyadenylation signal sequence
2. The 3 end of the cut pre-mRNA undergoes
enzymatic addition of 20 to 200 adenines through
the action of CPSF and PAP
69
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Steps of Polyadenylation, continued
5. After addition of the first 10 adenines, molecules of
poly-A-binding protein (PABII) join the adenine tail
and increase the rate of addition of adenines
70
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
71
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Functions of Polyadenylation
1. Facilitating transport of mature mRNA across the
nuclear membrane to the cytoplasm
2. Protecting the mRNA from degradation
3. Enhancing translation by enabling the ribosomal
recognition of mRNA
• Some eukaryotic transcripts (e.g., the histone genes) do
not undergo polyadenylation
72
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Pre-mRNA Intron Splicing
• Intron splicing requires great precision to remove
intron nucleotides accurately
• Errors in intron removal would lead to incorrect
protein sequences
• Roberts and Sharp shared the 1993 Nobel Prize for
their codiscovery of “split genes,” i.e., the presence
of intron and exon sequences
73
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Splicing Signal Sequences
• Specific short sequences define the junctions
between introns and exons
• The 5 splice site is at the 5 intron end and contains
a consensus sequence with an invariant GU
dinucleotide at the 5-most end of the intron
• The 3 splice site at the opposite end of the intron
has an 11 nucleotide consensus with a pyrimidine
rich region and a nearly invariant AG at the
3-most end
74
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
The Branch Site
• A third consensus region, called the branch site, is
20 to 40 nucleotides upstream of the 3 end of the
intron
• It is pyrimidine-rich and contains an invariant
adenine called the branch point adenine near the 3
end of the consensus
• Mutation analysis shows that all three consensus
sequences are required for accurate splicing
75
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
76
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Splicing
• Introns are removed from the pre-mRNA by an
snRNA-protein complex called the spliceosome
• Like molecular “workbench”
• The 5 splice site is cleaved first
• Then the 3 splice site is cleaved and the exon ends
are ligated together
77
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
ANIMATION: RNA Splicing
78
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Accurate Splicing
• Spliceosome components are recruited to 5 and 3
splice sites by SR proteins (pathfinders)
• SR proteins bind to sequences in exons called
exonic splicing enhancers (ESEs), and ensure
accurate splicing
• May play a roll in alternative intron splicing
79
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
80
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Coupling of Pre-mRNA Processing Steps
• Introns appear to be removed one by one, but not
necessarily in order
• The three steps of pre-mRNA processing are tightly
coupled
• The carboxyl terminal domain (CTD) of RNA
polymerase II functions as an assembly platform and
regulator of pre-mRNA processing machinery
81
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Gene Expression Machines
• Current models suggest that RNA pol II and an
array of pre-mRNA processing proteins function as
“gene expression machines”
• The proteins that carry out capping, intron splicing,
and polyadenylation associate with the CTD of pol II
• All three processes are carried out simultaneously
82
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
83
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
ANIMATION: RNA Processing Control
84
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Alternative Transcripts of Single Genes
• It is common for large eukaryotic genomes to
express more proteins than there are genes in the
genome
• For example, human cells produce over 100,000
distinct polypeptides but contain  22,000 genes
• Three transcription-associated mechanisms can
explain this
85
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
The Human Genome
• Humans produce more than 100,000 distinct
peptides
• Assumed 80,000 -100,000 genes
• We were wrong
• ~22,000 genes in humans
• What’s going on?
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Alternative pre-mRNA Processing
1. pre-mRNA can be spliced in alternative patterns in
different cell types
2. Alternative promoters can initiate transcription at
distinct start points
1. Different start point
3. Alternative localizations of polyadenylation can
produce different mature mRNAs
1. Different stop point
87
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Alternative Intron Splicing
• Alternative intron splicing: processing of identical
transcripts in different cells can lead to mature
mRNAs with different combinations of exons and
thus different polypeptides
• Approximately 70% of human genes are thought to
undergo alternative splicing
• It is less common in other animals and rare in plants
88
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
89
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Dscam
• The Drosophila Dscam gene has one of the most
complex patterns of alternative splicing
• Of the 24 exons, numbers 4, 6, 9, and 17 have
numerous alternative sequences
• More than 38,000 different polypeptides can be
produced through alternative splicing
• Not all of the possible arrangements are observed,
however
90
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
91
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Alternative Processing
• Alternative splicing is mainly controlled by variation
in SR proteins in different cell types
• Use of alternative promoters can occur when more
than one sequence upstream of a gene can initiate
transcription
• Alternative polyadenylation requires more than
one polyadenylation signal in a gene
• Alternative promoters of polyadenylation are
controlled by variable expression of regulatory
proteins in specific cell types
92
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Rat α-tropomyosin
• 14 exons
• With 4 alternates
• Two promoters
• Five polyadenylation sites
• Yields 9 mature mRNA
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
94
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Intron Self-Splicing
• RNAs can contain introns that catalyze their own
removal
• There are three categories of self-splicing introns,
group I, group II, and group III
• Group I introns were discovered in 1981 in the
laboratory of Thomas Cech
95
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Post-Transcriptional RNA Editing
• In the mid-1980s, RNA editing was uncovered; that
is responsible for post-transcriptional modifications to
the nucleotide sequence (and the protein produced)
of some mRNAs
• In one kind of RNA editing, uracils are added with the
assistance of a guide RNA (gRNA), which contains
a sequence complementary to the mRNA that it edits
• Editing may sometimes involve deletion of
uracils, too
96
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
97
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
Base Substitution
• A second type of RNA editing is base substitution,
frequently replacement of cytosine with uracil
• This has been identified in mammals, most land
plants, and some single-celled eukaryotes
98
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.
99
Genetics Analysis: An Integrated Approach
Copyright © 2012 Pearson Education Inc.