Transcript RNA polymerase

8
RNA Synthesis and
Processing
8 RNA Synthesis and Processing
• Transcription in Bacteria
• Eukaryotic RNA Polymerases and General
Transcription Factors
• Regulation of Transcription in Eukaryotes
• Chromatin and Epigenetics
• RNA Processing and Turnover
Introduction
Regulation of gene expression allows
cells to adapt to environmental
changes and is responsible for the
distinct activities of differentiated cell
types that make up complex
organisms.
Introduction
Transcription is the first step in gene
expression, and the initial level at which
gene expression is regulated.
In eukaryote cells, RNAs are then
modified and processed in various
ways.
Transcription in Bacteria
Studies of E. coli have provided the
model for subsequent investigations of
transcription in eukaryotic cells.
mRNA was discovered first in E. coli;
RNA polymerase was first purified from
E. coli.
Transcription in Bacteria
RNA polymerase catalyzes
polymerization of ribonucleoside 5′triphosphates (NTPs) as directed by a
DNA template, in the 5′ to 3′ direction.
Transcription initiates de novo (no primer
required) at specific sites—this is a
major step at which regulation of
transcription occurs.
Transcription in Bacteria
Bacterial RNA polymerase has five types
of subunits.
The σ subunit identifies the correct sites
for transcription initiation.
Most bacteria have several different σ’s
that direct RNA polymerase to different
start sites under different conditions.
Figure 8.1 E. coli RNA polymerase
Transcription in Bacteria
Promoter: gene sequence to which
RNA polymerase binds to initiate
transcription.
Promoters are six nucleotides long;
located at 10 and 35 base pairs
upstream of the transcription start site.
Consensus sequences are the bases
most frequently found in different
promoters.
Figure 8.2 Sequences of E. coli promoters
Transcription in Bacteria
Experiments show the functional
importance of –10 and –35 regions:
• Genes with promoters that differ from
the consensus sequences are
transcribed less efficiently.
• Mutations in these sequences affect
promoter function.
• The σ subunit binds to both regions.
Transcription in Bacteria
Initially, the DNA is not unwound
(closed-promoter complex).
The polymerase then unwinds 12–14
bases of DNA to form an openpromoter complex, allowing
transcription.
After addition of about 10 nucleotides, σ
is released from the polymerase.
Figure 8.3 Transcription by E. coli RNA polymerase (Part 1)
Figure 8.3 Transcription by E. coli RNA polymerase (Part 2)
Transcription in Bacteria
During elongation, polymerase maintains
an unwound region of about 15 bp.
High-resolution structural analysis shows
the β and β′ subunits form a crab-clawlike structure that grips the DNA
template.
A channel between these subunits
contains the polymerase active site.
Figure 8.4 Structure of bacterial RNA polymerase
Transcription in Bacteria
RNA synthesis continues until the
polymerase encounters a stop signal.
The most common stop signal is a
symmetrical inverted repeat of a GCrich sequence followed by seven A
residues.
Transcription in Bacteria
Transcription of the GC-rich inverted
repeat results in a segment of RNA that
forms a stable stem-loop structure.
This disrupts its association with the
DNA template and terminates
transcription.
Figure 8.5 Transcription termination
Transcription in Bacteria
Alternatively, transcription of some
genes is terminated by a specific
termination protein (Rho), which binds
extended segments of single-stranded
RNA.
Transcription in Bacteria
Most transcriptional regulation in
bacteria operates at initiation.
Studies of gene regulation in the 1950s
used enzymes involved in lactose
metabolism.
The enzymes are only expressed when
lactose is present.
Transcription in Bacteria
Three enzymes are involved:
• β-galactosidase cleaves lactose into
glucose and galactose.
• Lactose permease transports lactose
into the cell.
• Transacetylase inactivates toxic
thiogalactosides that are transported
into the cell along with lactose.
Figure 8.6 Metabolism of lactose
Transcription in Bacteria
Genes encoding these enzymes are
expressed as a single unit, an operon.
Two loci control transcription:
• o (operator), adjacent to
transcription initiation site
• i (outside the operon), encodes a
protein that binds to the operator
Figure 8.7 Negative control of the lac operon
Transcription in Bacteria
Mutants that don’t produce i gene product
express the operon even when lactose is
not available.
This implies that the normal i gene product
is a repressor, which blocks transcription
when bound to o.
When lactose is present in normal cells, it
binds to the repressor, preventing it from
binding to the operator, and the genes
are expressed.
Transcription in Bacteria
The lactose operon illustrates the central
principle of gene regulation:
• Control of transcription is mediated by
the interaction of regulatory proteins
with specific DNA sequences.
Transcription in Bacteria
Cis-acting control elements affect
expression of linked genes on the same
DNA molecule (e.g., the operator).
Other proteins can affect expression of
genes on other chromosomes (e.g., the
repressor).
The lac operon is an example of negative
control—binding of the repressor blocks
transcription.
Transcription in Bacteria
Negative control: the regulatory protein
(the repressor) blocks transcription.
Positive control: regulatory proteins
activate transcription.
Transcription in Bacteria
Example of positive control in E. coli:
Presence of glucose (preferred energy
source) represses expression of the lac
operon, even if lactose is also present.
This is mediated by a positive control
system: If glucose decreases, levels of
cAMP increase.
Transcription in Bacteria
cAMP binds to the regulatory protein
CAP (catabolite activator protein).
This stimulates CAP to bind to its target
DNA sequence upstream of the lac
operon.
CAP facilitates binding of RNA
polymerase to the promoter.
Figure 8.8 Control of the lac operon by glucose
Eukaryotic RNA Polymerases and General Transcription Factors
Transcription in eukaryotes:
• Eukaryotic cells have three RNA
polymerases that transcribe different
classes of genes.
• RNA polymerases must interact with
additional proteins to initiate and
regulate transcription.
Eukaryotic RNA Polymerases and General Transcription Factors
• Transcription takes place on
chromatin; regulation of chromatin
structure is important in regulating
gene expression.
Eukaryotic RNA Polymerases and General Transcription Factors
Eukaryotic RNA polymerases are
complex enzymes, with 12 to 17
different subunits.
They all have nine conserved subunits,
five of which are related to subunits of
bacterial RNA polymerase.
Yeast RNA polymerase II is strikingly
similar to that of bacteria.
Table 8.1 Classes of Genes Transcribed by Eukaryotic RNA Polymerases
Figure 8.9 Structure of yeast RNA polymerase II
Eukaryotic RNA Polymerases and General Transcription Factors
RNA polymerase II synthesizes mRNA
and has been the focus of most
transcription studies.
It requires initiation factors that (in
contrast to bacterial σ factors) are not
associated with the polymerase.
Eukaryotic RNA Polymerases and General Transcription Factors
General transcription factors are
proteins involved in transcription from
polymerase II promoters.
About 10% of the genes in the human
genome encode transcription factors,
emphasizing the importance of these
proteins.
Eukaryotic RNA Polymerases and General Transcription Factors
Promoters contain several sequence
elements surrounding the transcription
sites.
The TATA box resembles the –10
sequence of bacterial promoters.
Others include initiator (Inr) elements,
TFIIB recognition elements (BRE), and
downstream elements DCE, MTE, and
DPE).
Figure 8.10 Formation of a polymerase II preinitiation complex in vitro (Part 1)
Figure 8.10 Formation of a polymerase II preinitiation complex in vitro (Part 2)
Eukaryotic RNA Polymerases and General Transcription Factors
Five general transcription factors are
required for initiation of transcription in
vitro.
TFIID is composed of multiple subunits,
including TATA-binding protein (TBP)
and other subunits (TAFs) that bind to
the Inr, DCE, MTE, and DPE
sequences.
Eukaryotic RNA Polymerases and General Transcription Factors
Several other transcription factors
(TFIIB, TFIIF, TFIIE, and TFIIH) bind in
association with the RNA polymerase II
to form the preinitiation complex.
Figure 8.10 Formation of a polymerase II preinitiation complex in vitro (Part 3)
Eukaryotic RNA Polymerases and General Transcription Factors
Within a cell, additional factors are
required to initiate transcription.
These include Mediator, a protein
complex of 20+ subunits; it interacts
with both general transcription factors
and RNA polymerase.
Figure 8.11 RNA polymerase II/Mediator complexes and transcription initiation
Eukaryotic RNA Polymerases and General Transcription Factors
RNA polymerase I transcribes rRNA
genes, which are present in tandem
repeats.
Transcription yields a large 45S prerRNA, which is processed to yield the
28S, 18S, and 5.8S rRNAs.
Figure 8.12 Transcription of the ribosomal RNA gene
Eukaryotic RNA Polymerases and General Transcription Factors
Promoters of rRNA genes are
recognized by two transcription factors
that recruit RNA polymerase I to form
an initiation complex:
• UBF (upstream binding factor)
• SL1 (selectivity factor 1)
Eukaryotic RNA Polymerases and General Transcription Factors
Genes for tRNAs, 5S rRNA, and some of
the small RNAs are transcribed by
polymerase III.
They are expressed from three types of
promoters: TFIIIA, TFIIIB, and TFIIIC.
Figure 8.13 Transcription of RNA polymerase III genes
Regulation of Transcription in Eukaryotes
Eukaryotic DNA is packaged into
chromatin, which limits its availability
for transcription.
Non-coding RNAs and proteins regulate
transcription via modifications of
chromatin structure.
Regulation of Transcription in Eukaryotes
Many cis-acting sequences regulate
expression of eukaryotic genes.
These regulatory sequences have been
identified by gene transfer assays.
Regulation of Transcription in Eukaryotes
Gene transfer assays:
Regulatory sequences are ligated to
reporter genes that encode easily
detectable enzymes, such as firefly
luciferase.
The regulatory sequence directs
expression of the reporter gene in
cultured cells.
Figure 8.14 Identification of eukaryotic regulatory sequences
Regulation of Transcription in Eukaryotes
Two cis-acting promoters (TATA and GC
boxes) were identified in studies of the
herpes simplex virus gene that
encodes thymidine kinase.
cis-acting regulatory sequences are
usually located upstream of the
transcription start site.
Figure 8.15 A eukaryotic promoter
Regulation of Transcription in Eukaryotes
Enhancers: regulatory sequences
located farther away from the start site.
First identified in studies of the promoter
of virus SV40.
Activity of enhancers does not depend
on their distance from, or orientation
with respect to the transcription
initiation site.
Figure 8.16 The SV40 enhancer
Figure 8.17 Action of enhancers (Part 1)
Figure 8.17 Action of enhancers (Part 2)
Figure 8.17 Action of enhancers (Part 3)
Figure 8.17 Action of enhancers (Part 4)
Figure 8.17 Action of enhancers (Part 5)
Regulation of Transcription in Eukaryotes
Enhancers, like promoters, bind
transcription factors that then regulate
RNA polymerase.
DNA looping allows a transcription factor
bound to a distant enhancer to interact
with proteins associated with the RNA
polymerase/Mediator complex at the
promoter.
Figure 8.18 DNA looping
Regulation of Transcription in Eukaryotes
Example: an enhancer controls
transcription of immunoglobulin genes in
B lymphocytes.
Gene transfer experiments show that the
enhancer is active in lymphocytes, but
not in other cell types.
This regulatory sequence is partly
responsible for tissue-specific expression
of the immunoglobulin genes.
Regulation of Transcription in Eukaryotes
Enhancers usually have multiple
sequence elements that bind different
regulatory proteins that work together
to regulate gene expression.
The immunoglobulin heavy-chain
enhancer has at least nine sequence
elements that are protein-binding sites.
Figure 8.19 The immunoglobulin enhancer
Regulation of Transcription in Eukaryotes
Enhancers account for 10% or more of
human genomic DNA, emphasizing the
importance of these elements.
Many mutations linked to human
diseases affect enhancers rather than
protein-coding sequences.
In any given cell type, multiple
enhancers work together to regulate
individual genes.
Regulation of Transcription in Eukaryotes
Activity of any given enhancer is specific
for the promoter of its target gene.
Specificity is maintained partly by
insulators, which divide chromosomes
into independent domains and prevent
enhancers from acting on promoters
located in an adjacent domain.
Regulation of Transcription in Eukaryotes
Genomes are divided into discrete
chromosomal domains: topologically
associating domains (TADs).
Enhancers and promoters within a TAD
interact frequently with each other, but
only rarely with elements in other
domains.
The main protein that binds insulators in
vertebrates is CTCF.
Figure 8.20 Chromosomal domains and CTCF
Regulation of Transcription in Eukaryotes
Transcription factor binding sites have
been identified by various experiments:
Electrophoretic-mobility shift assay:
Radiolabeled DNA fragments are
incubated with a protein, then analyzed
by electrophoresis.
Migration of a DNA fragment is slowed by
a bound protein.
Figure 8.21 Electrophoreticmobility shift assay
Regulation of Transcription in Eukaryotes
Binding sites are usually short DNA
sequences (6–10 bp) and they are
degenerate:
• The transcription factor will bind to
the consensus sequence, but also to
sequences that differ from the
consensus at one or more positions.
Regulation of Transcription in Eukaryotes
Transcription factor binding sites are
shown as pictograms, representing the
frequency of each base at all positions
of known binding sites for a given
factor.
Figure 8.22 Representative transcription factor binding sites
Regulation of Transcription in Eukaryotes
Chromatin immunoprecipitation:
Cells are treated with formaldehyde to
cross-link transcription factors to the DNA
sequences to which they were bound.
Chromatin is extracted and fragmented.
Fragments of DNA linked to a
transcription factor can then be isolated
by immunoprecipitation.
Figure 8.23 Chromatin immunoprecipitation (Part 1)
Figure 8.23 Chromatin immunoprecipitation (Part 2)
Regulation of Transcription in Eukaryotes
One of the first transcription factors to be
isolated was Sp1, in studies of SV40
virus, by Tjian and colleagues.
Sp1 was shown to bind to GC boxes in
the SV40 promoter. This established
the action of Sp1 and also suggested a
method for purification of transcription
factors.
Key Experiment, Ch. 8, p. 279 (1)
Key Experiment, Ch. 8, p. 279 (2)
Regulation of Transcription in Eukaryotes
DNA-affinity chromatography:
Double-stranded oligonucleotides with
repeated GC box sequences are bound
to agarose beads in a column.
Cell extracts are passed through the
column. Sp1 binds to the GC box with
high affinity and is retained on the
column.
Figure 8.24 Purification of Sp1 by DNA-affinity chromatography
Regulation of Transcription in Eukaryotes
Transcriptional activators, like Sp1,
bind to regulatory DNA sequences and
stimulate transcription.
These factors have two independent
domains: one binds DNA, the other
stimulates transcription by interacting
with other proteins, such as Mediator.
Figure 8.25 Structure of transcriptional activators
Regulation of Transcription in Eukaryotes
Many transcription factors have now
been identified in eukaryotic cells.
About 2000 are encoded in the human
genome.
They contain many distinct types of
DNA-binding domains.
Regulation of Transcription in Eukaryotes
DNA binding domains:
1. Zinc finger domain: binds zinc ions
and folds into loops (“fingers”) that bind
DNA.
Steroid hormone receptors have zinc
fingers; they regulate gene transcription
in response to hormones such as
estrogen and testosterone.
Figure 8.26 Examples of DNA-binding domains (Part 1)
Regulation of Transcription in Eukaryotes
2. Helix-turn-helix domain: one helix
makes most of the contacts with DNA,
the others lie across the complex to
stabilize the interaction.
Homeodomain proteins are important in
regulation of gene expression during
embryonic development.
Figure 8.26 Examples of DNA-binding domains (Part 2)
Regulation of Transcription in Eukaryotes
3. Leucine zipper and helix-loop-helix
proteins: DNA-binding domains are
formed by dimerization of two
polypeptide chains.
Different members of each family can
dimerize with one another—
combinations can form an expanded
array of factors.
Figure 8.26 Examples of DNA-binding domains (Part 3)
Figure 8.26 Examples of DNA-binding domains (Part 4)
Regulation of Transcription in Eukaryotes
Activation domains of transcription
factors are not as well characterized.
They stimulate transcription by two
mechanisms:
• Interact with Mediator proteins and
general transcription factors
• Interact with coactivators to modify
chromatin structure.
Figure 8.27 Action of transcriptional activators
Regulation of Transcription in Eukaryotes
Gene expression is also regulated by
repressors, which inhibit transcription.
In some cases, they simply interfere with
binding of other transcription factors.
Other repressors compete with
activators for binding to specific
regulatory sequences.
Figure 8.28 Action of eukaryotic repressors (Part 1)
Regulation of Transcription in Eukaryotes
Active repressors have domains that
inhibit transcription via protein-protein
interactions.
These include interactions with specific
activator proteins, with Mediator
proteins or general transcription
factors, and with corepressors that act
by modifying chromatin structure.
Figure 8.28 Action of eukaryotic repressors (Part 2)
Regulation of Transcription in Eukaryotes
Transcription can also be regulated at
elongation.
Recent studies show that many genes
have molecules of RNA polymerase II
that have started transcription but are
stalled immediately downstream of
promoters.
Regulation of Transcription in Eukaryotes
The RNA polymerase II is poised to
continue in response to appropriate
signals.
Many genes on which poised
polymerases have been found are
regulated by extracellular signals or
function during development.
Regulation of Transcription in Eukaryotes
Following initiation, the polymerase
pauses within about 50 nucleotides due
to negative regulatory factors, including
NELF (negative elongation factor) and
DSIF.
Continuation depends on another factor:
P-TEFb (positive transcriptionelongation factor-b).
Figure 8.29 Regulation of transcriptional elongation (Part 1)
Figure 8.29 Regulation of transcriptional elongation (Part 2)
Chromatin and Epigenetics
Because eukaryotic DNA is packaged in
chromatin, chromatin structure is a
critical aspect of gene expression.
Histones can be modified several
ways—key mechanisms for regulating
gene expression.
Many histone modifications are stably
inherited when cells divide.
Chromatin and Epigenetics
The basic unit of chromatin is the
nucleosome: 147 bp of DNA wrapped
around two molecules each of histones
H2A, H2B, H3, and H4.
One molecule of histone H1 is bound to
the DNA as it enters the nucleosome
core particle.
Figure 6.17 Structure of a chromatosome (Part 1)
Repeat fig. 6.17 A here
Chromatin and Epigenetics
Chromatin limits availability of DNA for
transcription, affecting both
transcription factor binding and action
of RNA polymerase.
Actively transcribed genes are in
relatively decondensed chromatin,
which can be seen in polytene
chromosomes of Drosophila.
Figure 8.30 Decondensed chromosome regions in Drosophila
Chromatin and Epigenetics
Chromatin can be altered by histone
modifications and nucleosome
rearrangements.
Chromatin and Epigenetics
Histone acetylation:
Amino-terminal tail domains of core
histones are rich in lysine and can be
modified by acetylation.
Actetyl groups are added by histone
acetyltransferase (HAT) and removed
by histone deacetylase (HDAC).
Figure 8.31 Histone acetylation (Part 1)
Chromatin and Epigenetics
Acetylation neutralizes the positive
charge of lysine, relaxing chromatin
structure and increasing availability of
the DNA template for transcription.
Transcriptional activators and repressors
are associated with HAT and HDAC.
Figure 8.31 Histone acetylation (Part 2)
Chromatin and Epigenetics
Histones can also be modified by
methylation of lysine and arginine,
phosphorylation of serine, and addition
of small peptides (ubiquitin and SUMO)
to lysine.
These modifications occur at specific
amino acid residues in the histone tails.
Figure 8.32 Patterns of histone modification
Chromatin and Epigenetics
Histone modifications affect gene
expression by altering chromatin
properties, and by providing binding
sites for proteins that activate or
repress transcription.
Methylated H3 lysine-9 and -27 residues
are binding sites for proteins that
induce chromatin condensation and
formation of heterochromatin.
Chromatin and Epigenetics
Promoters and enhancers have distinct
chromatin features.
They are free of nucleosomes, and thus
accessible for binding transcription
factors.
These regions can be digested with with
DNase (DNase hypersensitive sites).
Figure 8.33 Chromatin at promoters and enhancers (Part 1)
Chromatin and Epigenetics
Flanking nucleosomes have different
histone modifications.
Example: Promoters have trimethylated
H3 lysine-4; enhancers have a
monomethylated form of this lysine.
Figure 8.33 Chromatin at promoters and enhancers (Part 2)
Chromatin and Epigenetics
Chromatin remodeling factors are
protein complexes that alter contacts
between DNA and histones.
They can reposition nucleosomes,
change conformation of nucleosomes,
or eject nucleosomes from the DNA.
Figure 8.34 Chromatin remodeling factors
Chromatin and Epigenetics
Following initiation of transcription,
elongation is facilitated by elongation
factors associated with the
phosphorylated C-terminal domain of
RNA polymerase II.
Elongation factors include histone
modifying enzymes and chromatin
remodeling factors.
Chromatin and Epigenetics
Modified histones serve as binding sites
for proteins that themselves catalyze
histone modifications.
Histone modifications can thus regulate
one another, leading to the stable
patterns of modified chromatin.
Chromatin and Epigenetics
This provides a mechanism for
epigenetic inheritance —transmission
of information that is not in the DNA
sequence.
Modified histones are transferred to both
progeny chromosomes where they direct
similar modification of new histones—
maintaining characteristic patterns of
modification.
Figure 8.35 Epigenetic inheritance of histone modifications (Part 1)
Figure 8.35 Epigenetic inheritance of histone modifications (Part 2)
Figure 8.35 Epigenetic inheritance of histone modifications (Part 3)
Chromatin and Epigenetics
Example: Some regulatory genes are
repressed in certain cells in developing
Drosophila; the repression is passed on
in subsequent divisions.
Repression results from methylation of
H3 lysine 27 by the Polycomb
proteins.
Chromatin and Epigenetics
One complex (PRC1) binds to
methylated H3 lysine 27.
The other complex (PRC2) methylates
H3 lysine 27.
As a result, methylation of H3 lysine 27
can spread to adjacent nucleosomes
and is maintained when cells divide.
Figure 8.36 Polychrome proteins
Chromatin and Epigenetics
DNA methylation is another mechanism
of epigenetic control of transcription:
Methyl groups are added at the 5-carbon
position of cytosines that precede
guanines (CpG dinucleotides).
This methylation is correlated with
transcriptional repression.
Figure 8.37 DNA methylation
Chromatin and Epigenetics
Methylation is common in transposable
elements; it plays a key role in
suppressing their movement.
DNA methylation is also associated with
transcriptional repression of
mammalian genes involved in
development and differentiation.
Chromatin and Epigenetics
DNA methylation is an important
mechanism for epigenetic inheritance.
Following DNA replication, an enzyme
methylates CpG sequences of a
daughter strand that is hydrogenbonded to a methylated parental
strand.
Figure 8.38 Maintenance of methylation patterns
Chromatin and Epigenetics
Methylation can be reversed by the TET
family enzymes, which catalyze
oxidation of 5-methylcytosine to 5formylcytosine and 5-carboxylcytosine.
These are excised and replaced by
cytosine via DNA repair.
Figure 8.39 Reversal of methylation
Chromatin and Epigenetics
DNA methylation plays a role in
genomic imprinting: expression of
some genes depends on whether they
come from the mother or the father.
Example: Gene H19 is transcribed only
from the maternal copy. It is methylated
during development of male, but not
female, germ cells.
Figure 8.40 Genomic imprinting
Chromatin and Epigenetics
Transcription can also be regulated by
noncoding RNA molecules:
• miRNAs (20–30 nucleotides) act by
the RNA interference pathway to
inhibit translation or induce
degradation of homologous mRNAs.
Chromatin and Epigenetics
• Long noncoding RNAs (lncRNAs)
(>200 nucleotides):
Form complexes with proteins that modify
chromatin and recruit these complexes
to their sites of transcription, thereby
regulating expression of neighboring
genes.
Chromatin and Epigenetics
In many cases, lncRNAs repress their
target genes by forming complexes with
Polycomb Repressive Complex 2
(PRC2).
Includes Xist lncRNA, which mediates X
chromosome inactivation in mammals
and several lncRNAs involved in
imprinting.
Figure 8.41 Action of lncRNAs (Part 1)
Chromatin and Epigenetics
lncRNAs can associate with different
chromatin-modifying enzymes and
function as repressors or activators.
Some lncRNAs act in trans by recruiting
chromatin-modifying complexes (e.g.,
PRC2) to distant target genes.
Many other lncRNAs regulate gene
expression in multiple ways.
Figure 8.41 Action of lncRNAs (Part 2)
RNA Processing and Turnover
Bacterial mRNAs are used immediately
for protein synthesis while still being
transcribed.
But they are an exception; most RNAs
must be processed in various ways.
Regulation of processing provides
another level of control of gene
expression.
RNA Processing and Turnover
Ribosomal RNAs of both prokaryotes
and eukaryotes are derived from a
single long pre-rRNA molecule.
In prokaryotes, this is cleaved to form
three rRNAs (16S, 23S, and 5S).
Eukaryotes have four rRNAs; 5S rRNA
is transcribed from a separate gene.
Figure 8.42 Processing of ribosomal RNAs
RNA Processing and Turnover
tRNAs also start as long precursors
(pre-tRNAs) in prokaryotes and
eukaryotes.
Processing of the 5′ end of pre-tRNAs
involves cleavage by the enzyme
RNase P.
RNase P is a ribozyme—an enzyme in
which RNA rather than protein
catalyzes the reaction.
Figure 8.43 Processing of transfer RNAs (Part 1)
Figure 8.43 Processing of transfer RNAs (Part 2)
RNA Processing and Turnover
Processing of the 3′ end of tRNAs
involves addition of a CCA terminus,
the site of amino acid attachment.
Bases are also modified at specific
positions. About 10% of the bases are
modified.
Figure 8.43 Processing of transfer RNAs (Part 3)
Figure 8.43 Processing of transfer RNAs (Part 4)
RNA Processing and Turnover
Some pre-tRNAs have introns that are
removed by splicing.
tRNA splicing is mediated by
conventional proteins such as
endonuclease, rather than catalytic
RNAs.
RNA Processing and Turnover
In eukaryotes, pre-mRNAs are
extensively modified before export from
the nucleus.
Throughout processing, transport,
translation, and degradation, mRNA
molecules are associated with proteins
to form messenger ribonucleoprotein
particles (mRNPs).
RNA Processing and Turnover
Transcription and processing are
coupled.
The C-terminal domain (CTD) of RNA
polymerase II plays a key role by
serving as a binding site for the
enzymes involved in mRNA
processing.
RNA Processing and Turnover
The 5′ end of the transcript is modified
by addition of a 7-methylguanosine
cap.
The 5′ cap stabilizes the RNA, and
aligns it on the ribosome during
translation.
Figure 8.44 Processing of eukaryotic messenger RNAs (Part 1)
Figure 8.44 Processing of eukaryotic messenger RNAs (Part 2)
RNA Processing and Turnover
At the 3′ end, a poly-A tail is added by
polyadenylation.
Signals for polyadenylation are the
hexanucleotide AAUAAA, and a GUrich downstream element.
Poly-A polymerase then adds about 200
adenines to form the poly-A tail.
Figure 8.45 Formation of the 3′ ends of eukaryotic mRNAs
RNA Processing and Turnover
Recognition of the polyadenylation
signal leads to termination of
transcription, cleavage, and
polyadenylation of the mRNA.
The RNA that has been synthesized
downstream of the site of poly-A
addition is degraded.
RNA Processing and Turnover
Introns (noncoding sequences) are
removed from pre-mRNA by splicing.
In mammals, most genes contain
multiple introns.
Splicing has to be highly specific to yield
functional mRNAs.
RNA Processing and Turnover
In vitro systems were developed to study
splicing:
A gene with introns is cloned adjacent to
a promoter for a bacteriophage RNA
polymerase.
Transcription of these plasmids produced
pre-mRNAs that, when added to nuclear
extracts of mammalian cells, were found
to be correctly spliced.
Figure 8.46 In vitro splicing
RNA Processing and Turnover
Splicing proceeds in two steps:
1. Cleavage at the 5′ splice site (SS) and
joining of the 5′ end of the intron to an
adenine within the intron (branch point).
The intron forms a loop.
2. Cleavage at the 3′ SS and
simultaneous ligation of the exons
excises the intron loop.
Figure 8.47 Splicing of pre-mRNA
RNA Processing and Turnover
Three sequence elements of premRNAs are important:
At the 5′ splice site, at the 3′ splice site,
and within the intron at the branch
point.
Pre-mRNAs contain similar consensus
sequences at each of these positions.
RNA Processing and Turnover
Splicing takes place in large complexes,
called spliceosomes, which have five
types of small nuclear RNAs
(snRNAs)—U1, U2, U4, U5, and U6.
The snRNAs are complexed with 6–10
protein molecules to form small
nuclear ribonucleoprotein particles
(snRNPs).
RNA Processing and Turnover
snRNAs were first identified in the late
1960s, but their function was unknown.
In 1979, Lerner and Steitz demonstrated
that snRNAs were present as RNAprotein complexes called snRNPs, and
suggested that they might function in
pre-mRNA splicing.
Key Experiment, Ch. 8, p. 303 (2)
RNA Processing and Turnover
First step in spliceosome assembly:
binding of U1 snRNP to the 5′ SS.
Recognition of 5′ SS involves base
pairing between the 5′ SS consensus
sequence and a complementary
sequence at the 5′ end of U1 snRNA.
Figure 8.48 Assembly of the spliceosome (Part 1)
Figure 8.48 Assembly of the spliceosome (Part 2)
Figure 8.49 Binding of U1 snRNA to the 5′ splice site
RNA Processing and Turnover
U2 snRNP then binds to the branch
point.
The other snRNPs join the complex and
act together to form the intron loop, and
maintain the association of the 5′ and 3′
exons so they can be ligated.
This is followed by excision of the intron.
RNA Processing and Turnover
snRNAs recognize consensus
sequences at the branch and splice
sites, and also catalyze the splicing
reaction.
Some RNAs can self-splice: they
catalyze removal of their own introns in
the absence of other protein or RNA
factors.
RNA Processing and Turnover
Two groups of self-splicing introns:
Group I—cleavage at 5′ SS mediated by
a guanosine cofactor.
Group II—cleavage of 5′ SS results from
attack by an adenosine nucleotide in
the intron.
Figure 8.50 Self-splicing introns (Part 1)
Figure 8.50 Self-splicing introns (Part 2)
RNA Processing and Turnover
Other splicing factors bind to RNA and
recruit U1 and U2 snRNPs to the
appropriate sites on pre-mRNA.
SR splicing factors bind to specific
sequences in exons and recruit U1
snRNP to the 5′ SS.
U2AF binds to pyrimidine-rich sequences
at the 3′ SS and recruits U2 snRNP to the
branch point.
Figure 8.51 Role of splicing factors in spliceosome assembly
RNA Processing and Turnover
Alternative splicing:
Most pre-mRNAs have multiple introns,
thus different mRNAs can be produced
from the same gene.
This is one way of controlling gene
expression, and increases the diversity
of proteins that can be encoded.
RNA Processing and Turnover
Sex determination in Drosophila is an
example of tissue-specific alternative
splicing.
Splicing of transformer mRNA is
regulated by the SXL protein, which is
only expressed in females.
SXL acts as a repressor that blocks
splicing factor U2AF.
Figure 8.52 Alternative splicing in Drosophila sex determination
RNA Processing and Turnover
The Dscam gene of Drosophila has four
sets of exons; one from each set goes
into the spliced mRNA in any
combination, yielding 38,016 possible
mRNAs.
Different forms of Dscam provide
neurons with an identity code essential
in establishing connections between
neurons for brain development.
Figure 8.53 Alternative splicing of Dscam
RNA Processing and Turnover
RNA editing: processing (other than
splicing) that alters the protein-coding
sequences of mRNAs.
It involves single base modification
reactions, such as deamination of
cytosine to uridine and adenosine to
inosine.
RNA Processing and Turnover
Editing mRNA for apolipoprotein B, which
transports lipids in the blood, results in
two proteins:
• Apo-B100, produced in the liver by
translation of unedited mRNA
• Apo-B48, produced in the intestine
from mRNA in which a C has been
changed to a U by deamination
Figure 8.54 Editing of apolipoprotein B mRNA
RNA Processing and Turnover
A-to-I editing: deamination of adenosine
to inosine.
Results in single amino acid changes in
ion channels and receptors on the
surface of neurons.
Mutants lacking the editing enzyme have
a variety of neurological defects.
RNA Processing and Turnover
Over 90% of pre-mRNA sequences are
introns, which are degraded in the
nucleus after splicing.
Processed mRNAs are protected by
capping and polyadenylation, but the
unprotected ends of introns are
recognized and degraded by enzymes.
RNA Processing and Turnover
Defective mRNAs can also be degraded.
Nonsense-mediated mRNA decay
eliminates mRNAs that lack complete
open-reading frames.
When ribosomes encounter premature
termination codons, translation stops
and the defective mRNA is degraded.
RNA Processing and Turnover
Ultimately, RNAs are degraded in the
cytoplasm.
Levels of any RNA are determined by a
balance between synthesis and
degradation.
Rate of degradation can thus control
gene expression.
RNA Processing and Turnover
rRNAs and tRNAs are very stable in
both prokaryotes and eukaryotes.
This accounts for the high levels of these
RNAs (greater than 90% of all RNA) in
cells.
RNA Processing and Turnover
Bacterial mRNAs are rapidly degraded;
most have half-lives of 2–3 minutes.
Rapid turnover allows a cell to respond
quickly to changes in its environment,
such as nutrient availability.
RNA Processing and Turnover
In eukaryotes, mRNA half-lives vary; less
than 30 minutes to 20 hours.
Short-lived mRNAs code for regulatory
proteins, levels of which can vary rapidly
in response to environmental stimuli.
mRNAs encoding structural proteins or
central metabolic enzymes have long
half-lives.
RNA Processing and Turnover
Degradation of eukaryote mRNAs is
initiated by shortening the poly-A tails.
Rapidly degraded mRNAs often contain
specific AU-rich sequences near the 3′
ends—binding sites for proteins that
can either stabilize them or target them
for degradation.
Figure 8.55 mRNA degradation
RNA Processing and Turnover
These RNA-binding proteins are
regulated by extracellular signals, such
as growth factors and hormones.
Degradation of many mRNAs is
regulated by miRNAs, which stimulate
degradation as well as inhibit
translation.