26| RNA Metabolism

© 2017 W. H. Freeman and Company

 CHAPTER 26
RNA Metabolism

Goals:
• Learn Transcription: DNA-dependent synthesis of
RNA
• Learn Capping and splicing: RNA processing
• Learn RNA-dependent synthesis of RNA and DNA
 Overview of RNA Types
All RNA is made by transcription. There are many types of RNA produced by
transcription.

o Messenger RNAs (mRNA) are coding RNAs. mRNAs carry information contained
within DNA to the ribosome, where they direct the sequence of amino acids during
protein synthesis, according to the mRNA sequence and the 'genetic code'. The
sequence of codons (nucleotide triplets) in an mRNA determines the amino acid
sequence in a protein. Some mRNAs contain cis regulatory elements, such as
riboswitches, in the untranslated regions (either 3' or 5' UTRs).
o Ribosomal RNAs (rRNA) are structural and catalytic components of the ribosome, the
large RNA-protein assembly where protein is synthesized in all living systems. In the
ribosome, amino acids are transfered from tRNAs to a nascent (growing) polypeptide
chain, with the amino acid sequence controlled by the mRNA. The peptidyl
transferase center, which is the catalytic site of the ribosome, is all rRNA. So
technically the ribosome is a ribozyme, not a protein enzyme.
o Transfer RNAs (tRNA) are RNAs that become covalently linked to amino acids
(activating the amino acids). tRNAs contain anti-codons that interact with condons on
mRNAs. tRNAs transfer amino acids to a nascent polypeptide chain in the ribosome.
The covalent linkage between a given amino acid and the correct (cognate) tRNA is
catalyzed by a specific aminoacyl-tRNA synthetase (one for each amino acid). The
aminoacyl-tRNA synthetases establish and enforce the genetic code.
o MicroRNAs (miRNAs) are ~22 nucleotides in length and are found only in eukaryotic
 Overview of RNA Types
All RNA is made by transcription. There are many types of RNA produced by
transcription.

o Ribosomal RNAs (rRNA) are structural and catalytic components of the ribosome, the
large RNA-protein assembly where protein is synthesized in all living systems. In the
ribosome, amino acids are transfered from tRNAs to a nascent (growing) polypeptide
chain, with the amino acid sequence controlled by the mRNA. The peptidyl
transferase center, which is the catalytic site of the ribosome, is all rRNA. So
technically the ribosome is a ribozyme, not a protein enzyme.
o Transfer RNAs (tRNA) are RNAs that become covalently linked to amino acids
(activating the amino acids). tRNAs contain anti-codons that interact with condons on
mRNAs. tRNAs transfer amino acids to a nascent polypeptide chain in the ribosome.
The covalent linkage between a given amino acid and the correct (cognate) tRNA is
catalyzed by a specific aminoacyl-tRNA synthetase (one for each amino acid). The
aminoacyl-tRNA synthetases establish and enforce the genetic code.
o MicroRNAs (miRNAs) are ~22 nucleotides in length and are found only in eukaryotic
cells (but not fungi, algae, etc). miRNAs bind to complementary sequences on target
mRNAs, usually resulting in down regulation and silencing of gene expression (mRNA
degradation & sequestering and translational suppression)
o Long non-coding RNAs (long ncRNAs, lncRNA, in eukaryotes) are transcripts (>200
nucleotides) that are not translated into protein. Only one-fifth of transcription across
the human genome is associated with protein-coding genes, there is four times more
 Overview of RNA Types
All RNA is made by transcription. There are many types of RNA produced by
transcription.

o Transfer RNAs (tRNA) are RNAs that become covalently linked to amino acids
(activating the amino acids). tRNAs contain anti-codons that interact with condons on
mRNAs. tRNAs transfer amino acids to a nascent polypeptide chain in the ribosome.
The covalent linkage between a given amino acid and the correct (cognate) tRNA is
catalyzed by a specific aminoacyl-tRNA synthetase (one for each amino acid). The
aminoacyl-tRNA synthetases establish and enforce the genetic code.
o MicroRNAs (miRNAs) are ~22 nucleotides in length and are found only in eukaryotic
cells (but not fungi, algae, etc). miRNAs bind to complementary sequences on target
mRNAs, usually resulting in down regulation and silencing of gene expression (mRNA
degradation & sequestering and translational suppression)
o Long non-coding RNAs (long ncRNAs, lncRNA, in eukaryotes) are transcripts (>200
nucleotides) that are not translated into protein. Only one-fifth of transcription across
the human genome is associated with protein-coding genes, there is four times more
long non-coding than coding RNA sequences. It is not exactly clear what all these
lncRNAs are doing
o Also: (1) Small nucleolar RNAs (snoRNAs) guide chemical modifications of other
RNAs, mainly ribosomal RNAs, transfer RNAs and small nuclear RNAs.
 Overview of RNA Types
All RNA is made by transcription. There are many types of RNA produced by
transcription.

o MicroRNAs (miRNAs) are ~22 nucleotides in length and are found only in eukaryotic
cells (but not fungi, algae, etc). miRNAs bind to complementary sequences on target
mRNAs, usually resulting in down regulation and silencing of gene expression (mRNA
degradation & sequestering and translational suppression).

o Long non-coding RNAs (long ncRNAs, lncRNA, in eukaryotes) are transcripts (>200
nucleotides) that are not translated into protein. Only one-fifth of transcription across
the human genome is associated with protein-coding genes, there is four times more
long non-coding than coding RNA sequences. It is not exactly clear what all these
lncRNAs are doing
o Also: (1) Small nucleolar RNAs (snoRNAs) guide chemical modifications of other
RNAs, mainly ribosomal RNAs, transfer RNAs and small nuclear RNAs.
 Overview of RNA Types
All RNA is made by transcription. There are many types of RNA produced by
transcription.

o Long non-coding RNAs (long ncRNAs, lncRNA, in eukaryotes) are transcripts (>200
nucleotides) that are not translated into protein. Only one-fifth of transcription across
the human genome is associated with protein-coding genes, there is four times more
long non-coding than coding RNA sequences. It is not exactly clear what all these
lncRNAs are doing.

o Also: (1) Small nucleolar RNAs (snoRNAs) guide chemical modifications of other
RNAs, mainly ribosomal RNAs, transfer RNAs and small nuclear RNAs.
 Overview of RNA Types
All RNA is made by transcription. There are many types of RNA produced by
transcription.

o Also: (1) Small nucleolar RNAs (snoRNAs) guide chemical modifications of other
RNAs, mainly ribosomal RNAs, transfer RNAs and small nuclear RNAs.
 Overview of RNA polymerases
RNA polymerase (RNAP) is an enzyme that produces RNA using
DNA as a template. RNAPs are essential to modern life and are
found in all living systems.

RNAPs are nucleotidyl transferases that initiate synthesis de novo

(do not required primers) and add ribonucleotides to the 3' hydroxyl
group of RNA molecules. The reaction is driven by release of PPi
(hydrolysis of PPi to Pi + Pi). Unlike DNA polymerase, RNAP can
initiate a new RNA strand without a primer.
Overview of RNA polymerases

Transcription “Bubble”
 Topology issues during elongation.

Figure 26-7
Overview of RNA polymerases

This image is insane.

No, magnesium is
gigantic (actual ionic
radius = 0.67 Å); it
not bigger than a
purine. Yes, all
phosphates are the
same size. And RNA
and NTP have a 2’
hydroxyl group.
 Transcription in E. Coli
Rapid Transcription Reinitiation

Facilitated recycling: Preinitiation intermediates on activated genes can persist.

The retention of transcription proteins at an actively transcribed gene increases the frequency
• The nucleoside triphosphates add to the the 3’ end
of subsequent transcription initiation

of the growing RNA strand.

• The growing chain is complementary to the template
strand in DNA.
• The synthesis is catalyzed by enzyme (RNA
http://wwwmgs.bionet.nsc.ru/mgs/gnw/trrd/db_schema.shtml

polymerase) and involves 2 Mg2+ ions for catalysis.

• RNA polymerase unwinds ~17bp of DNA and covers
about 35 bp of DNA.
 Overview of RNA Metabolism
(for •
differentiation,
Transcribed cell type,
fromdevelopment,
DNA exercise, diet, environmental stimuli, food sources,
(for
aging) differentiation, cell type, development, exercise, diet, environmental stimuli, food sources,
aging)
– Transcription is tightly regulated in order to control the concentration
of each protein.
Specificity factors (sigma factors) alter the specificity of RNA polymerase for promoters (DNA
Specificity factors (sigma factors) alter the specificity of RNA polymerase for promoters (DNA
• Ribozymes
sequences).
sequences).
– Being
Repressors bind tomainly single stranded,
DNA Operators, manytoRNA
that are close molecules
or overlapping thecan fold into
promoter region.
Repressors bind to DNA Operators, that are close to or overlapping the promoter region.
compact structures with specific functions.
General transcription factors position RNA polymerase at the start of a protein-coding
General
sequence transcription
– and
Some
thenRNA factors position
molecules
release RNA
can act
the polymerase toaspolymerasetheat
catalysts
transcribe the start of aoften
(ribozymes),
mRNA. protein-coding
using
sequence and then release the polymerase to transcribe the mRNA.
metal ions as cofactors such as the group I introns.
Activators enhance interactions between the RNA polymerase and particular promoters.
Activators enhance interactions between the RNA polymerase and particular promoters.
• Processing of mRNAs
Enhancers (far away from promoter) are bound by activators that loop the DNA, bringing a
Enhancers
specific (far to
away
– Splicing
promoter from promoter)
- elimination
the initiation are boundjoining
of introns;
complex. by activators that loop the DNA, bringing a
of exons
specific promoter to the initiation complex.
– Poly-adenylation of the 3’ end
Silencers are DNA sequences that, when bound by particular transcription factors, can inhibit
Silencers are DNA sequences that, when bound by particular transcription factors, can inhibit
– Capping the 5’ end
transcription
transcription
Epigenetics by DNA methylation & histone modification.
Epigenetics by DNA methylation & histone modification.
Go the CRC: Change your Gene Expression:
SIRTs regulate metabolism at the transcriptional level and more directly
control the activity of metabolic enzymes.
 Features of Transcription

• RNA polymerase binds to a sequence called

promoter to begin transcription.
– primer not required
• The growing end of new RNA temporarily base-pairs
with DNA template for ~8 bp and inserts 50-90nt/sec.
• The DNA duplex unwinds, forming a “bubble” of ~17
bp.
• The RNA Pol generates positive supercoils ahead,
negative supercoils behind, relieved by
topoisomerases.
 Bacterial RNA Polymerase is a large
enzyme at least six subunits
• RNA polymerase holoenzyme has
five core subunits of 2’ plus a
sixth called .
• See Figure 26-4.
• RNA Pol lacks 3’  5’-exonuclease,
no proofreading, so it has a high
error rate of 1/104–1/105.
• RNA polymeras binds to promoter
regions to initiate transcription.
 Bacterial RNA Polymerase is a large
enzyme at least six subunits

• Two  subunits function in assembly and binding to

UP (upstream promoter) elements.
– See slide on promoters and Fig. 26-5
• The  subunit is the main catalytic subunit.
• The ’ subunit is responsible for DNA binding.
• The  subunit directs enzyme to the promoter.
– Each class of RNA pol holoenzymes has a different  subunit.
• The  appears to protect the polymerase from
denaturation.
 TABLE 26-1 The Seven σ Subunits of E. coli
Holoenzyme
σ subunit Kd (nM) Molecules/cella ratio (%)a Function
σ70 0.26 700 78 Housekeeping
σ54 0.30 110 8 Modulation of cellular nitrogen levels
σ38 4.26 <1 0 Stationary phase genes
σ32 1.24 <10 0 Heat shock genes
σ28 0.74 370 14 Flagella and chemotaxis genes
σ24 2.43 <10 0 Extracytoplasmic functions; some heat shock functions
σ18 1.73 <1 0 Extracytoplasmic functions, including ferric citrate transport
Source: Data from H. Maeda et al., Nucleic Acids Res. 28:3497, 2000.
Note: σ factors are widely distributed in bacteria; the number varies from a single σ factor in Mycoplasma genitalium to 63 distinct σ factors
in Streptomyces coelicolor.
aApproximate number of each σ subunit per cell and the fraction of RNA polymerase holoenzyme complexed with each σ subunit during

exponential growth. The numbers change as growth conditions change. The fraction of RNA polymerase complexed with each σ subunit
reflects both the amount of the particular subunit and its affinity for the enzyme.
 Common Features of Promoters in E. Coli

• There are two consensus sequences at −10 (TATAAT)

and −35 (TTGACA) for  subunit binding.
– called TATA sequences
• An A-T−rich upstream promoter element between
−40 and −60 binds the  subunit.
• A-T−rich sequences promote strand separation.
• These sequences govern efficacy of RNA Pol binding
and therefore affect gene-expression level.
• Nucleotides before the first nucleotide of the RNA
molecule are considered “upstream” and given
negative values.
Examples of E. coli Promoter
Regions
A promoter is a DNA sequence from -1 to around -40 (i.e., on the 5’ side of the sense
strand of gene, upstream of the start site). RNAP binds to the Pribnow box during
initiation which is the first region where base pairs are disrupted. The Pribnow box
(TATAAT) is the most conserved part of the promoter.
Expression of genes (or operons) is controlled partly by various σ factors that
recognize the -10 to -35 region. Different σ factors recognize different sequences. The
α subunit recognizes an upstream element (-40 to -70 base pairs, TTGACA) of the
DNA.
Steps in bacterial transcription

 (1) Initiation
Starts at the +1 position, usually a purine. RNAP does not require a
primer. Initiation involves a DNA Promoter, Transcription Factors,
DNA Helicases, RNAP, Activators and Repressors. RNAP binds very
tightly to the promotor (KD=10-14M ). The promoter region (from -9
to +2) is melted (strands are separated), forming a transcription
bubble.

Eukaryotes use six General Transcription Factors (GTFs) to form a

PreInitiation Complex (PIC). Transcription Factor IID (TF IID)
contains TATA Box binding Protein (TBP). TBP binds to the TATA
box but also to TATA-less promoters.
 Initiation of Transcription

• RNA Pol binds to promoter with the  factor

bound.
– It creates a closed complex (DNA is not
unwound).
• An open complex forms.
– The region from ~−10 to ~+2 unwinds.
• RNA Pol moves away from the promoter.
–  is replaced by the protein NusA.
Transcription Initiation and Elongation
in E. Coli
Steps in transcription

(2) Promoter clearance

After initiation the RNAP has a tendency to release truncated RNA
transcripts (abortive initiation). Abortive initiation continues to occur
until the σ factor rearranges and is released.

Eukaryotes: RNAP II clears the promoter and leaves behind some of

the GTFs including TFIID (the second RNAP II complex can
reinitiate more quickly than the first).
Steps in transcription

(3) Elongation
RNA polymerase traverses the DNA template (antisense) strand, and
following the rules of Watson-Crick complementarity with the
antisense strand, creates an RNA copy of the sense (coding) strand.
Polymerization is processive (without dissociation). Transcripts can
be thousands or even millions of nucleotides. The rate of
polymerization is around 50 nucleotides/second, slower than
replication. The error rate of transcription is around 1 in 4000. RNA
polymerase traverses the template strand from 3' → 5'.
Polymerization occurs in the 5' → 3' direction. The resulting RNA
transcript is a copy of the sense (coding, non-template) strand, except
that thymines are replaced with uracils, and deoxyriboses are
replaced by riboses. A second RNAP can quickly reinitiate from the
same site.
Eukaryotes: The C-Terminal Domain (CTD) of RpbI is
phosphorylated and binds to a six protein complex called Elongator.
Steps in transcription

 (4) Termination

Transcription terminates at specific sites. Bacteria use two strategies for

termination.

In Rho-independent termination, the newly synthesized RNA molecule

forms a G-C-rich stem-loop followed by a run of A's and U's. It seems
the stem loop causes the RNAP to pause and ultimately to dissociate.

In the "Rho-dependent" termination, a protein "Rho" destabilizes the

interaction between the template and the mRNA, thus releasing the
newly synthesized mRNA from the elongation complex.

Eukaryotes lack specific transcription termination sites. The 3’ ends of

the transcription product are heterogeneous, but are cleaned up by
processing before translation (3’ poly A tails are added).
 Two Types of Termination in E. Coli

1. -independent
- characterized by three
U’s near the 3’ end of
the transcript
– self-complementary
regions in transcript
form a hairpin 15−20
nt before the 3’ end
• makes the RNA Pol
pause, dissociate
 Rho-independent termination at a GC-rich
palindrome.

Figure 26-9a
 Two Types of Termination in E. Coli

2. -dependent
– common CA-rich
sequence called a rut site
(Rho utilization element)
–  protein processes until
termination site reached
–  protein is a helicase,
binds to rut site
rho

The Rho factor recognizes ~70 nucleotides in the RNA product of

transcription. The rho utilization site in the transcript (rut) is single-
stranded, rich in cytosine and poor in guanine. Several rho binding
sequences are known.
Nature. 2007 Jul 12;448(7150):157-62. Epub 2007 Jun 20.

Structural basis for transcription elongation by bacterial RNA polymerase.

Vassylyev DG, Vassylyeva MN, Perederina A, Tahirov TH, Artsimovitch I.

Source

Department of Biochemistry and Molecular Genetics, University of Alabama at

Birmingham, Schools of Medicine and Dentistry, 402B Kaul Genetics Building, 720
20th Street South, Birmingham, Alabama 35294, USA. dmitry@uab.edu

Abstract: The RNA polymerase elongation complex (EC) is both highly stable and
processive, rapidly extending RNA chains for thousands of nucleotides.
Understanding the mechanisms of elongation and its regulation requires detailed
information about the structural organization of the EC. Here we report the 2.5-A
resolution structure of the Thermus thermophilus EC; the structure reveals the post-
translocated intermediate with the DNA template in the active site available for pairing
with the substrate. DNA strand separation occurs one position downstream of the
active site, implying that only one substrate at a time can specifically bind to the EC.
The upstream edge of the RNA/DNA hybrid stacks on the beta'-subunit 'lid' loop,
whereas the first displaced RNA base is trapped within a protein pocket, suggesting a
mechanism for RNA displacement. The RNA is threaded through the RNA exit
channel, where it adopts a conformation mimicking that of a single strand within a
double helix, providing insight into a mechanism for hairpin-dependent pausing and
 In these two views (the
next page shows the view
across the RNAP main
channel), the RNAP core
enzyme structure in the
ttEC is colored from grey
(less than 2 Å) to green
(more than 6 Å) according
to the deviation in the
positions of the Ca atoms of
the holoenzyme (PDB ID
2A6H).
The rest of the color
scheme is
grey, RNAP;
red, DNA template;
blue, DNA non-template;
yellow, RNA; magenta,
The high-affinity Mg2+ ion,
is also shown.

The green-grey coloring scheme shows

changes in structure compared to the
holoenzyme, which is the initiation complex.
The holoenzyme contains , which enables
promoter-specific transcription initiation.
from Vassylyev, Nature, 2007
 Here is a second
view of the structure
of the T. thermophilus
RNAP EC (ttEC) at
2.5 Å resolution in
which the core
enzyme (subunit
composition 2’)
is bound to 14 base
pairs (bp) of the
downstream DNA, 9
bp of the RNA/DNA
hybrid and seven
single-stranded
nucleotides of the
displaced RNA
transcript

from Vassylyev, Nature, 2007

In all organisms, transcription by DNA-dependent RNA polymerases
(RNAPs) can be divided into three mechanistically and structurally
distinct stages: initiation, elongation and termination. During initiation,
RNAP recognizes a promoter, unwinds DNA near the start site (register i
+ 1) and begins RNA synthesis, using NTPs as both a primer and the
substrates. Initiation is characterized by multiple rounds of abortive
synthesis during which RNAP synthesizes and releases short RNA
products. Once RNAP has made an RNA molecule 13–15 nucleotide
long, in which 7–9 nucleotides pair with the DNA template strand in an
RNA/DNA hybrid, the transcription complex undergoes promoter
clearance and makes a transition to the elongation phase.

Elongation is highly processive: the elongation complex (EC) is capable

of the uninterrupted synthesis of RNA chains thousands of nucleotides
long, yet becomes abruptly destabilized at terminators that demarcate
the RNA end, in many cases with single-nucleotide precision. The
interplay between processive synthesis, transient halting at numerous
'roadblocks' and RNA release depends on the intricate network of
interactions between RNAP, the nucleic acid signals and/or auxiliary
transcription factors within the EC.
from Vassylyev, Nature, 2007
RNA Polymerase and Generation of
Positive Supercoils
 Template versus Coding Strand
• DNA template strand – serves as template for RNA
polymerase
• DNA coding strand – the nontemplate strand; has
the same sequence as the RNA transcript
***Regulatory sequences are listed by the coding
strand sequence.
 Both DNA Strands can Code for
Proteins
• Coding information may be on either strand (“top” or
“bottom”).
• Adenovirus is one of the causative agents of the common
cold.
• Adenovirus has a linear genome.
• Each strand codes for a number of proteins.
 Eukaryotes RNA Polymerases
• RNA polymerase I synthesizes pre-ribosomal RNA (precursor
for 28S, 18S, and 5.8 rRNAs).
• RNA polymerase II is responsible for synthesis of mRNA.
– very fast (500–1000 nucleotides/sec)
– specifically inhibited by mushroom toxin -amanitin
– can recognize thousands of promoters (See Fig. 26-8)
• RNA polymerase III makes tRNAs and some small RNA
products.
• Plants appear to have RNA polymerase IV that is responsible
for the synthesis of small interfering RNAs.
• Mitochondria have their own RNA polymerase.
 Transcription Factors

Transcription factors bind to specific DNA sequences and

regulate transcription of specific genes. Transcription factors
activate and/or repress wide repertoires of genes in a
combinatorial fashion. Some transcription factors are at the
end signal transduction pathways that change TF (the
subcellular localization, phosphorylation state, etc). Post-
translational modifications to transcription factors in the
cytosol can cause them to translocate to the nucleus where
they can interact with enhancers.
 Features of Some Promoters Recognized
by Eukaryotic RNA Polymerase II

• Consensus sequence TATA(A/T)A(A/T)(A/G) ~−30

• Inr sequence (Initiator) ~+1
• Specific regulatory sequences farther upstream
 Eukaryotic mRNA Transcription
Involves Many Proteins
• Relies on protein-protein contacts
– many highly conserved transcription factors
• RNA Pol II is well-studied.
– large complex of 12 subunits
• Some subunits have some structural homology to
bacterial RNA polymerase.
– has carboxy-terminal domain of highly conserved
repeats
 TABLE 26-2 Proteins Required for Initiation of Transcription at the RNA Polymerase II (Pol II) Promoters of
Eukaryotes
Number of different
Transcription protein subunits Subunit(s) Mra Function(s)
Initiation
Pol II 12 7,000–220,000 Catalyzes RNA synthesis
TBP (TATA-binding protein) 1 38,000 Specifically recognizes the TATA box
TFIIA 2 13,000, 42,000 Stabilizes binding of TFIIB and TBP to the promoter
TFIIB 1 35,000 Binds to TBP; recruits Pol II–TFIIF complex
TFIIDb 13–14 14,000–213,000 Required for initiation at promoters lacking a TATA box

TFIIE 2 33,000, 50,000 Recruits TFIIH; has ATPase and helicase activities
TFIIF 2–3 29,000–58,000 Binds tightly to Pol II; binds to TFIIB and prevents binding of
Pol II to nonspecific DNA sequences
TFIIH 10 35,000–89,000 Unwinds DNA at promoter (helicase activity); phosphorylates
Pol II (within the CTD); recruits nucleotide-excision repair
proteins
Elongationc
ELLd 1 80,000
pTEFb 2 43,000, 124,000 Phosphorylates Pol II (within the CTD)
SII (TFIIS) 1 38,000
Elongin (SIII) 3 15,000, 18,000,
110,000
aMr reflects the subunits present in the complexes of human cells. Some components differ somewhat in size in yeast.
bThe presence of multiple copies of some TFIID subunits brings the total subunit composition of the complex to 21–22.
cThe function of all elongation factors is to suppress the pausing or arrest of transcription by the Pol II–TFIIF complex.
dName derived from eleven-nineteen lysine-rich leukemia. The gene for ELL is the site of chromosomal recombination events frequently associated with acute myeloid

leukemia.
 Assembly of RNA Polymerase II at
Promoter
• Initiated by TATA-binding protein (TBP) with the
promoter
-TBP is part of multisubunit complex TFIID.
• Other proteins include TFIIB, TFIIA, TFIIF, TFIIE and
TFIIH.
• Helicase activity in TFIIH unwinds DNA at the
promoter.
• Kinase activity in TFIIH phosphorylates the
polymerase at the CTD (carboxy-terminal domain),
changing the conformation and enabling RNA Pol II to
transcribe.
Transcription Using RNA Pol II

CDT: 52 repeats of
Tyr-Ser-Pro-Thr-Ser-Pro-Ser
 Elongation and Termination

• After 60-70nt, TFIIE is released followed by

TFIIH.
• Elongation factors bound to RNA Pol II enhance
processivity and coordinate posttranslational
modifications.
– Some elongation factors are bound to the
phosphorylated CTD.
• For termination, Pol II is dephosphorylated.
• Regulation is complex
 TFIIH and Repair

• Transcribed genes are more actively repaired

than silent genes are.
• It may partly be explained that TFIIH also has
a role in nucleotide-excision repair (NER).
– recruits the NER complex at a lesion
• Genetic repair diseases are associated with
TFIIH defects:
– xeroderma pigmentosum and others
end of transcription
 Processing of mRNA − Overview
• Dozens of proteins coordinate are involved in RNA
transport to ribosomes.
• An unprocessed, newly synthesized RNA molecule is
referred to as a primary transcript.
• Processing includes:
– splicing out introns and rejoining any exons for a continuous
sequence
– adding a 5’-cap
– adding a 3’-poly(A) tail
– degradation
Maturation of mRNA in Eukaryotes
 5’ Capping

• 5’ end of mRNA is capped with 7-methylguanylate (5’-5’

linkage),
• Capping enzyme complex (CEC) binds to RNA polymerase
II before transcription starts.
• As soon as the 5′ end of the new transcript emerges from
RNA polymerase II, it is capped by the CEC
• Capping
– Regulates nuclear export
– Prevents degradation by nucleases
– Promotes translation,
– Promotes 5′ proximal intron excision
 The 5’-Cap
• 7-methylguanosine links to 5’-
end via 5/,5’-triphosphate link.
– formed with a molecule of GTP
– may include additional methyl-
ations at 2’OH groups of next two
nucleotides following the cap
– methyl groups derive from S-
adenosylmethionine (SAMe)
• See Figure 26-12.
• Protects RNA from nucleases
• Forms a binding site for ribosome
 Splicing: Introns Are Found in many Genes

• Most genes in vertebrates, and some in yeast,

a few bacteria have introns.
• Exons are usually <1,000 bp in length.
• Introns are 50−700,000 bp in length.
– 1,800 bp avg
• Some genes have dozens of introns.
– The human genome has more than 200,000
introns spread across ~20,000 genes.
 Four Classes of Introns
• Group I and group II introns are self-splicing.
– require no additional proteins or ATP
– in nuclear, mitochondrial, and chloroplast genomes
– differ mainly in the splicing mechanism
– found in some bacteria
• Spliceosomal introns are spliced by enormous complexes called
spliceosomes.
– the most common introns
– frequent in protein-coding regions of eukaryotic genomes
• tRNA introns are spliced by protein-based enzymes.
– Primary transcript is cleaved by endonuclease.
– Exons are joined by ATP-dependent ligase.
 Splicing of Group I Introns
• 3’-OH of free guanosine (GMP,
GDP, etc.) used as a
nucleophile
• Attacks phosphodiester bond
between U and A at the end of
the intron
• Releases first portion of U-
ending exon
• 3’-OH of the U-ending exon
then attacks the 5’-end of the
other piece of exon to rejoin
the pieces
Nucleophilic Attack of Guanoisine 3’-OH on
UA of Exon-Intron Interface in Group I Intron
 Group II Introns

• The nucleophile is a 2’-OH

of an A residue within the
intron.
• After the first cleavage, the
second (rh side) piece forms
a lariat-like intermediate
with a 2’-5’-phosphodiester
bond.
• Other features are similar
to group I introns.
 Spliceosomal Introns Are Removed via
a Large Complex Called a Spliceosome

• The spliceosome is made up of snRNPs

(“snurps” for small nuclear ribonuclear
proteins).
– snRNP RNA is called snRNA (for small nuclear
RNA), which are 100−200 nt long.
• five snRNAs known in eukaryotes (U1, U2, U4, U5, U6)
• GU at 5’-end and AU at 3’-end usually mark
sites of splicing.
• Only eukaryotes have spliceosomes
 Binding of U1 snRNP and U2 snRNP
• Contain RNA regions complementary to mRNA
• U1 helps define the 5’-splice site.
• U2 binds near the 3’-end of the intron.
– creates a bulge that partly displaces and activates an A to be a
better nucleophile
– This A forms the 2’5’-phosphodiester bond of the lariat-like
intermediate.
 The 3’ Poly(A) Tail
• protects mRNA from enzymatic degradation
• aids in transcription termination,
• aids in export of the mRNA from the nucleus,
• important in translation,
• useful for molecular biology
• RNA Pol II synthesizes RNA beyond the
cleavage signal sequence.
– The cleavage signal is bound by an
endonuclease and a polyadenylate
polymerase bound to CTD.
• An endonuclease cleaves RNA 10−30 nt
downstream to highly conserved AAUAA.
• polyadenylate polymerase synthesizes
80−250 nt of A.
Overview of mRNA Processing
 Degradation of Cellular mRNAs
• The RNA lifetime is one means of gene regulation.
– Each gene product is needed for a unique amount of time .
• Half-lives vary from seconds to hours.
– rate of synthesis : rate of degradation
– steady state – rates are equal and balanced
– typical vertebrate mRNA ~3 hrs
– ~10 turnovers per cell generation
– shorter (~1.5 mins) half-lives for bacterial mRNAs
• Degradation occurs via ribonucleases.
• Hairpin structures in bacterial mRNA can extend half-life.
• In eukaryotes, the 5’cap and 3’ poly (A) tail aid in the stability
of the mRNA.
 Processing of tRNA and rRNA
• Bases are modified in posttranscription reactions.
– pseudouridine ()
– thiouridine
– dihydrouridine, and so on
 Processing of tRNA and rRNA
• rRNAs and tRNAs are cleaved from longer precursors.
MicroRNAs Function in Gene Regulation
• MicroRNAs (miRNAs):
– short noncoding RNAs of ~22 nucleotides
– bind to specific regions of mRNA to alter gene
expression
• assist in cleaving the mRNAs
• or block the mRNA from translation
– About 1,500 human genes encode miRNAs and 1
or more affect the expression of MOST protein-
coding genes!
– Synthesized from larger precursors
• processed by two endoribonucleases, Drosha and Dicer
 Ribozymes: Are RNA Molecules That
Catalytically Cleave RNA
also: the ribosome is a ribozyme
• Cleave themselves or another RNA
• 3-D structure integral to function
• Inactive if denatured
• Follows Michaelis-Menton kinetics
– saturable
– active site
– measureable KM
– competitively inhibited
– nucleophilic attack of sugar OH on phosphate
(transesterification) followed by phosphodiester bond
hydrolysis
 Examples of Well-characterized
Ribozymes
– Self-splicing group I introns
• Example: 26S rRNA precursor from protozoan
Tetrahymena
– Hammerhead ribozyme
• cleaves RNA of virusoids (circular RNAs that are
replicated by plant viruses)
• so named because 2 structure looks like head of
hammer
– RNase P
• cleaves precursors to tRNAs by recognizing the shape of
the pre-tRNA and the CCA sequence
 Ribozymes
Hammerhead Group I – rRNA intron from
Tetrahymena
 RNA-Dependent Synthesis of
Nucleic Acids
• Most DNA and RNA is synthesized
from a DNA template; however,
viruses do not fit with the norm.
• Retroviruses have genomes of
ssRNA and the enzyme reverse
transcriptase.
– virus enters host cell
– reverse transcriptase makes DNA
from the RNA
• It then degrades the RNA from the DNA-
RNA hybrid and replaces it with DNA.
• DNA can then be incorporated into host
DNA.
Retroviral Infection of a Mammalian Cell
and Integration into Host Chromosome
 Retroviruses Typically Contain Three Genes
Plus a Long Terminal Repeat

• gag (group associated antigen)

– encodes a long polypeptide that is cleaved into six smaller
proteins that make up viral core
• Pol (polypeptide)
– encodes protease that cleaves the long polypeptide,
reverse transcriptase, and an integrase to insert DNA into
host genome
• env
– encodes viral envelope
• Long terminal repeat (LTR) facilitates integration of
virus genome into host DNA
 Structure and Gene
Products of an
Integrated Retroviral
Genome
 Reverse Transcriptases Catalyze
Three Reactions
1. RNA-dependent DNA synthesis
2. RNA degradation
3. DNA-dependent DNA synthesis
• contain Zn2+, like DNA Pol
• use a primer of tRNA
• lack 3’  5’-proofreading, like RNA Pol
- make reverse transcriptase error-prone
- explains high rate of virus mutation/evolution (1: 20,000 nt)
 Some Retroviruses Cause Cancer

• Some retroviruses contain an oncogene.

– Example: The Rous sarcoma virus has the src
gene.
• Src for sarcoma, a cancer of bone, fat, muscle, etc. (vs.
cancer of epithelial cell origin)
• encodes a non-receptor tyrosine kinase, an enzyme
that affects cell division
 HIV Retrovirus Causes AIDS
• The HIV genome has genes for killing the host (mostly T
lymphocytes).
– results in suppression of immune system
• HIV-encoded reverse transcriptase is unusually error prone.
– complicates push for vaccine
– at least one error per replication, so potentially no two viral RNAs
alike
 Pharmaceutical Targets for HIV
(Antiretroviral Drugs)
• Reverse transcriptase inhibitors
– nucleotide or nucleoside analogs
– drug names ending in “dine” or “sine”:
• zidovudine (AZT), didanosine (Videx), and so on
• Protease inhibitors
– since proteases are used in cleaving proteins for
packaging into new viral particles
– drug names ending in “avir”:
• indinavir, saquinavir, and so on
 Retrotransposons in Eukaryotes Have
Similarities to Retroviruses
• Retrotransposons are mobile genetic
elements in eukaryotes.
– encode an enzyme with homology to reverse
transcriptase of retroviruses
– move between positions via RNA intermediates
• using their enzyme to make DNA from RNA
– unlike bacterial transposons that move directly
from DNA to DNA
 More About Retrotransposons

• Lack the env gene, so don’t form viral

particles
• Examples: Ty element in yeast and Copia
element in Drosophila
 Support for the RNA World Hypothesis
1. Synthesis of adenine from cyanide is simple; most enzyme
cofactors contain adenosine and it is the most abundant
nucleotide constituent.
2. Catalytic RNAs exist and there might have been a self-replicating
RNA.
3. Ribozymes can catalyze ester and amide bond formations, SN2
reactions, metalation of porphyrins, and C-C bond formation.
4. Peptide bond formation requires RNA molecules.
5. Many nonliving entities (RNA viruses, retoviruses,
retrotransposons) exist and can parasitize living cells.
6. Is there a ribozyme capable of directing its own synthesis?
 Chapter 26: Summary
In this chapter, we learned that:
• RNA polymerase synthesizes RNA using a strand of DNA as a
template and nucleoside triphosphates as substrates
• the primary RNA transcript in eukaryotes requires processing
before it becomes messenger RNA
• the processing involves capping the 5’ end with methylguanosine
to stabilize the RNA molecule
• the processing involves splicing out introns
• some introns have an amazing ability to carry out their own
splicing