Transcription and RNA

Processing
106 CENG
W10
Prokaryotic Transcription
Binding of RNA Polymerase to a
Promoter Sequence
• Promoter: The site right upstream of transcription
start site. This is where RNA polymerase binds.
Promoter region is not transcribed. It is more like a
landing pad. Transcription starts at transciption
start site.
• The terms upstream and downstream refer to
sequences located toward the 5′ or 3′ end of the
transcription unit, respectively
• The promoter is upstream of the transcribed
sequence
Essential Sequences in a Typical
Bacterial Promoter
• Right upstream of transcription start site (-10, -35), there
are evolutionarily conserved sequences.
• These sequences are bound by special proteins called sigma
factors (σ). Sigma factors direct RNA polymerase to
promoters and help with its binding.
• There are different sigma factors for different sets of genes:
Heat shock genes are controlled by a unique sigma factor.
This allows for fast and coordinated control on gene
expression.
RNA Polymerase Subunits
• Bacterial cells have a single kind of RNA polymerase
(unlike eukaryotes) that synthesizes all major
classes of RNA

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 Prokaryotic transcription

• Sigma factors recognize the start

sites of genes and direct RNA
polymerase there.
• Upstream of the transcription start
sites of genes are called
promoters.
• The DNA template unzips/melts and
sigma factor leaves. RNA
polymerase synthesizes the mRNA.
• In prokaryotes, transcription
termination happens in specific
DNA sites called terminators.
Elongation of the RNA Chain
• Transcription elongation continues as RNA
polymerase moves along the DNA molecule
• The RNA is elongated in the 5′ to 3′ direction, with
each new nucleotide added to the 3′ end
• As the polymerase moves along the DNA strand,
the double helix ahead of the polymerase is
unwound, and the DNA behind it is rewound into a
double helix
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RNA Proofreading
• When an incorrect nucleotide is incorporated, the
polymerase backs up slightly and the incorrect
nucleotide and the previous one are removed, in a
process called RNA backtracking
• This is RNA proofreading; occasional errors in RNA
molecules are not as critical as errors in DNA
replication

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Termination of RNA Synthesis
• Elongation of the RNA chain proceeds until the RNA
polymerase copies a sequence called the
termination signal. Together with the help of
termination proteins, RNA polymerase stops
transcription and releases the RNA.
Prokaryotic gene expression
Eukaryotic Transcription
 Transcription in Eukaryotic Cells Has Additional
Complexity Compared with Prokaryotes

• Eukaryotic transcription involves the same stages (RNA

polymerase recruitment, transcription initiation,
elongation and termination) as prokaryotic, but there are
several important differences
• Three different RNA polymerases transcribe one or more
different classes of RNA
• Eukaryotic promoters are more varied than bacterial ones
• Newly forming RNA molecules undergo RNA processing,
chemical modification during and after transcription
Eukaryotic Transcription
• Similar to bacterial sigma factors, in eukaryotic cells
there are specialized proteins that can recognize
specific DNA sequences in promoters and help
recruit RNA polymerase to these genes. These
proteins are called transcription factors.
• Transcription factors (TFs) regulate expression of a
group of genes. This allows for fast and coordinated
gene expression response.
RNA Polymerase I, II, and III Carry Out
Transcription in the Eukaryotic Nucleus

• There are three RNA polymerases in the nucleus,

designated RNA polymerases I, II, and III
• These differ in their location in the nucleus and the
types of RNA they synthesize
The RNA Polymerases
• RNA polymerase I, in the nucleolus, synthesizes an
RNA molecule that is a precursor for four types of
rRNA
• RNA polymerase II is found in nucleoplasm and
synthesizes mRNA.
• RNA polymerase III, in the nucleoplasm,
synthesizes a variety of small RNAs, including tRNA,
and the 5S rRNA

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 Eukaryotic transcription
• Similar to prokaryotic transcription but
much more complex.
• Promoter (transcription start) regions
contain specific DNA sequences that
are targeted by transcription factors.
• TATA box is bound by TATA-binding
protein (TBP).
• Other transcription initiation factors get
recruited to promoters, along with RNA
polymerase.
• Upon melthing of DNA double helix,
RNA polymerase starts transcribing.
Additional Control Elements
• Core promoters are capable of driving only a basal
(low) level of transcription
• Additional short DNA sequences upstream of
promoters (ENHANCER) improve the promoter’s
efficiency
Transcription is mediated by long distance sequences (enhancers)
and numerous proteins that bind promoters and enhancers
Other Proteins Needed for
Transcription
• Besides general transcription factors and RNA
polymerase II, several other kinds of proteins are
needed
• Some open chromatin structure to make DNA
accessible to RNA polymerase; others are
regulatory factors that bind upstream control
elements and recruit coactivator proteins

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 Elongation, Termination, and RNA Cleavage Are
Involved in Completing Eukaryotic RNA
Synthesis
• After initiation, RNA polymerases move along the DNA
and synthesize a complementary RNA
• Termination is governed by signals that differ for each
type of RNA polymerase
• For RNA polymerase II, transcripts are cleaved at a
specific site before transcription ceases
• The cleavage site is 10–35 nucleotides downstream of a
AAUAAA sequence in the RNA

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https://www.hhmi.org/biointeractive/dna-transcriptio
n-advanced-
detail
mRNA Processing
 Messenger RNA Processing in Eukaryotes
Involves Capping, Addition of Poly(A), and
Removal of Introns
• Most bacterial RNA is synthesized in a form that is ready
for translation with no need for processing
• Because there is no nuclear membrane, bacterial
transcripts are translated as they are transcribed
Transcription and Translation in
Eukaryotes
• Eukaryotic transcripts must be exported from the
nucleus to be translated
• Substantial processing occurs in the nucleus before
export
• Primary transcripts are often very long, 2000–
20,000 nucleotides (pre-mRNA).
Eukaryotic Transcripts

• Pre-mRNAs are processed by removal of sequences

and addition of 5′ caps and 3′ tails
• The C-terminal domain of one of the subunits of
RNA polymerase II acts as a platform for protein
complexes involved in processing. Therefore most
of RNA processing occurs during the synthesis of
mRNA during transcription
• mRNA processing in eukaryotes:
• 5’ cap
• 3’ polyA tail
• Splicing (removal of introns)
5′ Caps and 3′ Poly(A) Tails
• Eukaryotic mRNAs have a modified
nucleotide called the 5′ cap, and the
3′ ends have a long stretch of
adenines called the poly(A) tail
• The 5′ cap is a guanosine that is
methylated at position 7 of the
purine ring
• It is bound to the RNA molecule by a
5′→5′ linkage rather than the usual
3′→5′ bond
Roles of the 5′ Cap
• The 5′ cap is added soon after transcription is
initiated (so: during transcription)
• The cap contributes to mRNA stability by protecting
the RNA from nucleases
• The cap also plays a role in positioning the RNA on
the ribosome for initiation of translation
The Poly(A) Tail
• The poly(A) tail ranges from 50 to 250 nucleotides
long and is added by the enzyme poly(A) polymerase
• A signal for addition of the poly(A) tail, AAUAAA, is
located just upstream of the polyadenylation site, and
a GU- or U-rich element is located downstream of it
• The cleavage site of polymerase II transcripts is also
the site for addition of a poly(A) tail
• This is a string of adenine nucleotides added to the 3′
end of most eukaryotic mRNAs
Function of the Poly(A) Tail
• The poly(A) tail protects the mRNA from nuclease
attack; the length of the tail influences stability
• It is also required for export of the transcript to the
cytoplasm
• It may also help ribosomes recognize and bind
mRNAs
R Looping
• Single-stranded RNA and double-stranded DNA that
have the same nucleotide sequence are hybridized
under conditions that favor RNA-DNA hybrid
molecules
• DNA that is not hybridized to RNA is displaced as a
single-stranded loop, easily observed with electron
microscopy
• Some genes contained multiple loops

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Exons and Introns
• Sequences that appear in the final mRNA are called
exons
• Introns are present in most protein-coding genes of
multicellular eukaryotes
• The size and number of introns varies considerably
Spliceosomes Remove Introns from
Pre-mRNA
• The process of removing introns and joining the
exons is RNA splicing
• About 15% of inherited human diseases involve
splicing errors; such errors lead to incorrect protein
products
The Spliceosome
• Intron removal is catalyzed by large molecular
complexes called spliceosomes, consisting of five
types of RNA and many proteins
• Spliceosomes assemble on transcripts from a group
of smaller RNA-protein complexes called snRNPs
(small nuclear ribonuclearprotein complexes), each
containing one or two snRNA molecules (small
nuclear RNA)
• Most introns are destroyed without serving any
obvious function
DO NOT MEMORIZE THE DETAILS!
Just observe the main scheme

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Alternative Splicing
• The presence of introns allows each gene’s pre-
mRNA molecule to be spliced in multiple ways,
leading to production of multiple protein products
or a related protein.
• This alternative splicing is possible via mechanisms
allowing certain splice sites to be activated or
skipped
• Alternative splicingt can lead to tissue specific
protein products with variable
functions/structures/activities.
The C-Terminal Domain of RNA Polymerase
II Coordinates RNA Processing
• Many RNA processing events occur
cotranscriptionally (during transcription)
mRNA stability
Most mRNA Molecules Have a
Relatively Short Life Span
• Most mRNA molecules have a high turnover rate
(rate at which molecules are degraded and
replaced)
• It is measured in terms of half-life, the time
required for 50% of the molecules to degrade
• mRNA molecules of eukaryotes have half-lives of
several hours to a few days; in bacteria, the half-
lives are usually only a few minutes
The Abundance of mRNA Allows
Amplification of Genetic Information
• mRNA can be synthesized again and again from a
piece of template DNA, providing an opportunity
for amplification of genetic information
• For example, the haploid genome of the silkworm
has only one copy of the fibroin gene, but about 104
copies of the mRNA are transcribed from two
copies of the gene
How can you compare distinct
cell types?
Gene expression profiling=
Transcriptome profiling
1. Which RNA type in the cell would you be
interested in?
2. How can you isolate mRNA from cells?
3. How can you identify ALL of the mRNAs of a
particular cell type?
4. What percentage of genes do you think are
differentially expressed between two cells?
5. Can you directly sequence RNA similarly to DNA
sequencing (remember the human genome
project for DNA sequencing)?
RNA-sequencing
• This is a misnomer: RNA cannot be sequenced
directly. It needs to be converted to DNA first. Do
you remember any enzyme that can go from RNA
to DNA (reverse of the central dogma)?
• Step 1: mRNA isolation
• Step 2: Fragmentation of the RNA to 100-200
bases: Current sequencing techniques have
capabilities to read only this much!
RNA-sequencing
• Step 3: Conversion to complementary DNA (cDNA).
Is the cDNA of an mRNA molecule exactly the same
as its gene sequence?
• Step 4: Addition of adapter sequences (=stretches
of known DNA sequence) to enable sequencing
• Step 5: sequence
• Step 6: Analyze (how?)
RNA-sequencing
How to analyze RNA-seq data?
• First step after getting the sequence reads is that
you need to figure out which genomic region those
fragments come from.
• Step 1: Align to genome. Which regions in the
transcribed gene would you not observe any
sequencing reads?
RNA-seq analysis
• Step 2: Assembling mRNAs from fragmented cDNA
reads.
RNA-seq analysis
• Step 3: Quantifying the relative amount of each
mRNA: RPKM: Reads per kilobase million
RNA-seq analysis
• Step 4: Normalization and Identification of genes
expressed differently in the analyzed cell types

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4794996/
Variations in mRNA processing in different cell types
can be observed through RNA-seq

• Alternative splicing
• Alternative polyadenylation (Guest lecturer next
week: Assoc. Prof. Elif Erson Bensan)