Lecture 6 (biol3600) transcription m rna processing- winter 2012 pw

Benjamin A. Pierce

GENETICS
A Conceptual Approach
FOURTH EDITION

CHAPTER 13
Transcription

© 2012 W. H. Freeman and Company

Chapter 13 Outline

• 13.1 RNA, Consisting of a Single Strand of Ribonucleotides,
Participates in a Variety of Cellular Functions, 352

• 13.2 Transcription Is the Synthesis of an RNA Molecule from a
DNA Template, 354

• 13.3 The Process of Bacterial Transcription Consists of Initiation,
Elongation, and Termination, 359

• 13.4 Eukaryotic Transcription Is Similar to Bacterial Transcription
but Has Some Important Differences, 364

DNA TRANSCRIPTION
“Asthma, cancer, heart disease, immune disorders and viral infections
are seemingly disparate conditions. Yet they turn out to share a
surprising feature. All arise to a great extent from overproduction or
underproduction of one or more proteins, the molecules that carry out
most reactions in the body.” Sci. Amer. 1995

13.1 RNA Consisting of a Single Strand of Ribonucleotides,
Participates in a Variety of Cellular Functions

• The structure of RNA
– Primary structure
– Secondary structure

13.2 Transcription Is the Synthesis of an RNA
Molecule from a DNA Template

• The template:
– The transcribed strand: template strand.

13.2 Transcription Is the Synthesis of an RNA
Molecule from a DNA Template

– The transcription unit
• A promoter
• RNA-coding sequence
• Terminator

Transcription, mRNA processing, and translation
Instructions  Product

• DNA serves as the instruction manual for making proteins

• Going from instructions to product occurs in 3 main steps:

1. Transcription
- The information contained in the DNA is copied
into a complementary strand of RNA (ribonucleic acid)
- This RNA copy is called messenger RNA or mRNA
- Why is transcription necessary? (why copy the info)
- Location issues
- DNA is too valuable
2. mRNA editing
- The mRNA copy needs to be modified (cleaned-up)
before it leaves the nucleus
- Does not occur in prokaryotic cells
3. Translation
- Ribosomes read the mRNA and make a protein

Transcription
Activation of transcription

• Transcription is gene specific
- Each gene provides instructions for a single protein (usually)
- When turned on, only that one gene is transcribed (unlike DNA rep)

• What tells a cell to start transcribing a specific gene?
- Different signals for different genes in different cell types
1) Constituitive expression – Some genes are transcribed continuously
independent of cell health or environmental conditions
- Usually seen for those proteins that are needed 100% of the time
- Example: ATP synthase, actin, tubulin

2) Regulated expression
- Allows for much tighter control of gene
expression
Y YY PM
- Signal from outside or inside the cell
starts the whole process
- Often involves one or more signal
transduction pathways

Prokaryotic transcription
Initiation – Finding the gene
• All genes (eukaryotic and prokaryotic) have a unique region of DNA sequence
upstream of (before) the transcriptional start site that serves as a binding site for
the RNA polymerase enzyme (makes RNA)
- These regions are called promoters

promoter
promoter

RNA pol

• General functions of promoters:
1. Provides specificity – Tells the cell where the gene of interest is
- This is critical – transcribing the wrong gene could be deadly!!
- Each gene has a unique promoter sequence

2. Tell RNA polymerase where to start (they are usually at the beginning)
- Analogy: Front cover of a book
- No promoter (or mutated promoter), no transcription

3. Indicate which strand will be transcribed and the direction of transcription

Initiation – Finding the gene
• Although each promoter is unique,
most share a few common sequence elements called consensus sequences
- These common elements are evolutionarily conserved
- These common elements are extremely important for transcription
- If mutated, transcription does not take place

• Prokaryotic promoters share 2 main types of consensus sequences

- TATA box (aka Pribnow box) – found 10 nucleotides
upstream from the start site
(-10-15), sequence is usually TATAAT

- TTGACA – found 35 nucleotides upstream
from start (-35).
 Exact sequence and spacing can vary!!

• RNA polymerase enzyme makes direct contact
with the promoter at these consensus sequences

• Unique seq. within and around promoters help determine rates of transcription

Initiation – Binding of RNA polymerase to the promoter

• Prokaryotic cells only produce 1 type of RNA polymerase
- Produces all of the mRNA, tRNA, and rRNA in the cell

• Prokaryotic RNA polymerase structure:
- 2 alpha (α), 1 beta (β), 1 beta prime (β'), one omega ( ) β ασ
and 1 sigma (σ) subunit
α β'
- β, β’, α subunits interact and form the CORE ENZYME
- Together they have the basic catalytic function of producing new RNA

- σ factor controls binding to the promoter (PROMOTER RECOGNITION)
- After initial binding has been successful, the σ factor comes off the core
- ANALOGY: σ factor is the general, the core enzyme is the troops
- Bacteria make many different types of sigma factors
- Different sigma factors recognize different promoter sequences
- Examples: σ70 – controls transcription of general purpose genes
σ54 – controls transcription of genes only during nitrogen
starvation

Concept Check 2

What binds to the −10 consensus sequence found
in most bacterial promoters?

a. The holoenzyme (core enzyme + sigma factor)
b. The sigma factor alone
c. The core enzyme alone
d. mRNA

Initiation and elongation

• Once it binds to the specific promoter, RNA polymerase positions itself over the
transcriptional start site
- Chooses the start site based on its distance from the consensus sequences
- No specific “start signal” exists

• RNA pol unwinds the DNA near the start site (transcription bubble created)

• RNA pol reads 1st ~5-10 DNA nucleotides and brings in complimentary RNA
nucleotides (remember: U inserted across from A)
- Connects them via phosphodiester bonds
- Only transcribes one strand
- Moves 5’3’ (slowly initially)

• RNA polymerase changes shape after ~10 nucleotides
a) Cause it to lose its attraction for the consensus sequences
- Allows it to begin moving along the gene
b) Cause the sigma factor to fall off http://www.youtube.com/watch?v=WBcS3fKfbxs

 Once free, it moves quickly along the rest of the gene (5’3’)

Termination

• RNA pol continues producing a complimentary RNA molecule until it transcribes
a terminator signal (part of the sequence)
- ANALOGY: Not like a stop light. More like spikes on the road

• Two major prokaryotic transcriptional terminator signals exist
- Both must:
a) Slow down the RNA polymerase
b) Weaken the interaction between the DNA and RNA in the bubble

• Properties of the two major prokaryotic terminator signals
1. Rho-independent terminators
- The DNA sequence contains an inverted repeat followed
by a string of 6 adenines
- Following their transcription, the inverted repeats form
Hydrogen bonds with each other within the RNA and form
a hairpin loop
- Loop formation causes the RNA pol to pause
- The pause combined with weak hydrogen bonding between
6 straight A-U pairs cause the RNA to totally fall off of the
DNA template

Termination

• (… two major prokaryotic terminator signals)
2. Rho-dependent terminators
- Also contain inverted repeats that cause hairpin
loop formation in the new RNA
- Slows down the polymerase

- RNA contains a binding site for the Rho protein
- Rho moves towards the 3’ end

- Catches up to the RNA polymerase and unzips
the RNA from the DNA
- Acts as a RNA/DNA helicase

 These do not contain adenine rich region after
the inverted repeat

 Nearly all bacterial genes have one of these terminators

13.4 Eukaryotic Transcription Is Similar to
Bacterial Transcription but Has Some
Important Differences
• Transcription and nucleosome structure
– Chromatin modification before transcription.
• Promoters:
– Basal transcription apparatus
– Transcriptional activator proteins
– RNA polymerase II – mRNA synthesis
– Core promoter TATA box TATAAAA, -25 to -
30 bp, binded by transcription factors.

13.4 Eukaryotic Transcription Is Similar to
Bacterial Transcription but Has Some
Important Differences
• Transcription and nucleosome structure
– Chromatin modification before transcription.
• Promoters:
– Regulatory promoter
• Variety of different consensus sequences may be
found in the regulatory promoters.
– Fig. 13.16
– Enhancers:
– Polymerase I and polymerase III promoters.

Eukaryotic transcription
Activation and Initiation

• Eukaryotic transcription follows the same basic steps of prokaryotic transcription
- Activation, initiation, elongation, termination
- Major differences exist in how they accomplish the above steps

• General features of transcriptional activation is similar in prokaryotic and
eukaryotic cells
- Some genes are constituitively expressed, others are regulated
- The exact nature of the signals that activate transcription can be very different
- Example: Adding testosterone to a bacterial cell would likely do nothing

• Initiation – Making the DNA accessible
- Eukaryotic DNA is tightly coiled around proteins called
histones (and some nonhistone proteins)
- DNA is negatively charged (phosphate groups),
histones are very positively charged
- Every 146 base pairs are wrapped around
a histone octamer (8 histone proteins in a complex)
- Each group is called a nucleosome

Initiation – Making the DNA accessible

• Histones cont.
- To initiate transcription, DNA in the area of the gene has to be
loosened/unwound from histones and other proteins

• DNA is freed from histones in 2 major ways:
1) Histone acetylation
- Enzymes called histone acetyl transferases (HATs) add an acetyl group
(CH3CO) to histones (nucleophylic attack)
- This neutralizes their positive charge and causes
them to lose their attraction for DNA
- Occurs only in the area to be transcribed
(it is specfic!)
 Deacetylases remove acetyl groups after
transcription

2) Chromatin remodeling
- Some proteins move nucleosomes around (freeing up
the DNA) without directly modifying histones

Interferon - JAK/STAT Pathway

IFN

JAK JAK
STAT 1
P
P PP
Y Y

STAT 1

STAT 1
P
P
STAT 1


STAT 1
P
P
STAT 1

STAT 1
P Accessory TF
P motifs
TATA
GAS motif
STAT 1


Accessory TF
factors
STAT 1
P Accessory TF
P motifs
TATA
GAS motif
STAT 1

Initiation – Finding the gene (promoters and enhancers)

• Eukaryotic promoters
- Much more complex than those found in bacteria
- Some major consensus sequences (there are others):
1) TATA box
- Very similar to the prokaryotic TATA box, except the sequence
is slightly different (TATAAA) and it is located -25-30.

2) CAAT box - Located -70-80. Always contains either CAAT or CCAAT.
-Mutation of this region usually significantly lowers rate of transcription

3) GC box – Usually has the sequence GGGCGG and is typically found
~ -110 bps.

 Promoters can contain multiple copies of each of these consensus sequences
 The regions found between consensus sequences are unique in each
promoter (see next slide)

• Eukaryotic promoters
- Sequences between and around the three common consensus sequences are
different in each promoter

 Notice that each promoter is unique. Allows the cell to recognize it and
distinguish it from other promoters/genes

Initiation – Binding of RNA polymerases to the promoter

• Review of prokaryotic cells: A single type of RNA polymerase recognizes and
binds directly to consensus sequences in prokaryotic promoters

• Eukaryotic cells are a much more complicated – they have multiple RNA
polymerases and none of them recognize promoter sequences directly

• Eukaryotic cells have 3 RNA polymerases:
1) RNA polymerase I – Produces ribosomal RNA (rRNA)
2) RNA polymerase II – Produces messenger RNA (mRNA)
- All mRNA is made by RNA pol II (what we will discuss)
3) RNA polymerase III – Produces some types of rRNA and all tRNAs

 All function to transcribe a gene (DNA) into RNA (not just mRNA)

 All are much larger and more complex than the bacterial RNA polymerase
- Needs to be more complex because eukaryotic gene diversity
- Can have 20,000 different genes


• If eukaryotic RNA polymerases do not recognize promoter sequences directly,
how do they bind to the correct promoter?
 WITH THE HELP OF TRANSCRIPTION FACTORS!

• Transcription factors = Class of proteins that bind to DNA and help to recruit
RNA polymerase enzymes to promoters
(similar function as the __SIGMA_ in bacteria)
- As a result, they help to regulate eukaryotic transcription
- Two main classes
1) Basal transcription factors – Common set of proteins needed to get
transcription started (all promoters use these) – give low levels of
transcription
2) Regulatory transcription factors – Provide more specific transcriptional
control
 Either activate or repress transcription
above/below basal levels


• Binding of basal transcription factors to the promoter
(and recruitment of RNA pol II):
1) The TFIID complex binds to the
TATA box through its TBP subunit
- TBP = TATA-binding protein

2) This binding alters the shape of the
DNA and allows for binding of
TFIIA and TFIIB
- TFIIA = Stabilizes interaction between
TBP and the DNA
- TFIIB = Helps find the start site

3) RNA polymerase (escorted by TFIIF)
then comes in and interacts with the
preassembled complex

http://www.youtube.com/watch?v=JOBwqwxgJqc&feature=related


• Binding of basal transcription factors to the promoter
(and recruitment of RNA pol II):
4) Other factors then come in and help
RNA pol II to gain direct access to
the promoter
- TFIIH = Serves as a helicase to
separate the strands during
transcription

- Once this giant complex of proteins is
assembled on the TATA box, RNA pol II
will leave most of the other proteins
behind and start making mRNA

- The above events occur during all eukaryotic
gene transcription and provides just
basal levels of transcription
- When a cell wants more or less than this,
it will utilize regulatory transcription factors (discussed later)
http://www.youtube.com/watch?v=gZtGrsr8DMY

Elongation and termination

• Basic mechanism of elongation is essentially the same in
prokaryotic and eukaryotic cells
- Transcription bubble
- Ribonucleotides added onto the 3' end of the growing RNA

• Eukaryotic transcription termination
- Each type of RNA pol utilizes a different termination
mechanism
- None of them are as well characterized as termination
in bacteria
- RNA pol II = No clear termination signal
- Transcription continues well beyond the end of the gene
- However, there are characteristic termination sequences
(rich in As and Ts)
- Termination is coupled to mRNA processing (see later)

Concept Check 3

What is the difference between the core promoter
and the regulatory promoter?
a. Only the core promoter has consensus
sequences.
b. The regulatory promoter is farther upstream from
the gene.
c. Transcription factors bind to the core promoter;
transcriptional activator proteins bind to the
regulatory promoters.
d. Both b and c above

Benjamin A. Pierce

GENETICS
A Conceptual Approach
FOURTH EDITION

CHAPTER 14
RNA Molecules and RNA Processing

© 2012 W. H. Freeman and Company

Chapter 14 Outline

14.1 Many Genes Have Complex Structures, 376

14.2 Messenger RNAs, Which Encode the Amino
Acid Sequences of Proteins, Are Modified after
Transcription in Eukaryotes, 379

14.3 Transfer RNAs, Which Attach to Amino Acids,
Are Modified after Transcription in Bacteria and
Eukaryotic Cells, 389

Gene Organization
• The concept of colinearity and noncolinearity

The Concept of the Gene

• The gene includes DNA sequence that codes for
all exons, introns, and those sequences at the
beginning and end of the RNA that are not
translated into a protein, including the entire
transcription unit – the promoter, the RNA
coding sequence, and the terminator.

The structure of messenger RNA
• A mature mRNA contains 5′ untranslated region (5′
UTR, or leader sequence)
• Shine–Dalgarno sequence (ribosomal binding site)
• Protein coding region
• 3′ untranslated region

Regulation of transcription
Prokaryotic cells

• Transcriptional regulation in prokaryotic cells

1) Promoters and sigma factors
- As stated earlier, bacteria contain many different types sigma factors
- Different σ factors recognize different promoters
- Some σ factors allow for high levels of transcription, others only
promote low levels

- When a cell needs to transcribe specific genes, it
adds the appropriate σ factor to the core components  Sends it off

• Eukaryotic and prokaryotic cells have the ability to control how often specific
genes are transcribed (which would ultimately control protein levels)

- Example: Don’t need/want liver specific genes transcribed in brain cells

} Liver specific
genes
{

Gene Organization
• Introns
• Exons

Pre-mRNA processing

• The Addition of the 5′ cap:

• A nucleotide with 7-methylguanine; 5′-5′
bond is attached to the 5′-end of the RNA.

• The Addition of the poly(A) tail:

• 50 ~ 250 adenine nucleotides are added to
the 3′-end of the mRNA.

Eukaryotic mRNA processing
Capping of the mRNA

• Eukaryotic mRNA is modified prior to leaving the nucleus
1) Addition of a cap to the front (5') end
- The 5’ end of the growing mRNA is modified
very soon after the start of transcription

- Steps:
a) A guanine is added to the absolute 5’ end
via a 5’-5’ linkage to the 1st nucleotide
- Different from the normal 5’-3’ phosphodiester linkages

b) That guanine and the 1st few nucleotides are then methylated

c) Cap-binding proteins then attach to the cap
- Cap-binding proteins function to:
1) Protect the mRNA from RNases in the cytoplasm
2) Indirectly allow mRNA to attach to the
small ribosomal subunit

mRNA processing A
Addition of the poly A tail AAAAAAA

2) Addition of a poly-A tail to the back (3') end
- Most genes are transcribed beyond the coding sequence (sometimes
>1,000 extra)
- The extra sequence will be cut off and a poly A tail will be added

- Steps:
a) An enzyme (poly(A) polymerase) detects a consensus sequence
AAUAAA near the end of mRNA and cuts it ~25 nucleotides
downstream
b) The enzyme then adds 50-200 adenines to the cut end

- The string of adenines (poly A tail) function to:
1) Protect the 3’ end of the mRNA from
RNases
2) Allows cell to regulate mRNA stability
- Longer the poly A tail  Longer life span
3) Help in mRNA-ribosome binding

Pre-mRNA processing

• Nuclear organization
• Intron removal, mRNA processing, and transcription
take place at the same site in the nucleus.

• Minor Splicing

• Self-splicing introns happen in some rRNA genes in
protists and in mitochondria genes in fungi.

• Alternative processing pathways for processing pre-
mRNA.

mRNA processing
Removal of introns

3) Cutting out of INTRONS from the mRNA
- Most eukaryotic genes contain stretches
of noncoding sequence (introns) between
coding sequences (exons)
- Most genes split into good and useless parts

- Where did they come from?
- Retroviruses/transposons or mutated portion
of a former exon (lose part of the gene only)

- Some properties of introns:
a) Common in eukaryotes, rare in prokaryotes
b) More complex the organism, more complex/abundant the introns
c) Intron abundance and size vary per gene within a species
- Some genes have no introns, others have as many as 60

 THEY MUST BE REMOVED FROM THE mRNA!!!

mRNA processing
Removal of introns

- Introns are classified by how they are removed
- Some types of introns:
a) Group I and II introns
- Found in rRNA genes and a few bacterial genes
(generally small introns)
- Uses a molecule of guanosine to excise itself out
and “glue” the remaining exons together

b) Nuclear pre-mRNA introns
- Larger and more complex than Group I/II
- Require help from an enzyme complex called
the splicesome in order to remove its introns
- Usually contain consensus sequences
at the borders – attract the splicesome

mRNA processing
Removal of introns

- Alternative splicing of introns/exons can yield different proteins
- Some mRNAs can be spliced in different ways
- All introns removed, exons are differentially cut out/retained
- Different mRNAs  Different protein products (often called isoforms)
- The more exons, the more potential different isoforms
- Example
- Tropomyosin gene has 14 exons  10 different protein isoforms

ONE GENE≠ONE PROTEIN
opposite of what I told you previously

Concept Check 2

Alternative 3′ cleavage sites result in:

a. multiple genes of different length.
b. multiple genes of pre-mRNA of different length.
c. multiple mRNAs of different length.
d. all of the above.

Nuclear export of processed mRNA

• Processed eukaryotic RNAs will only exit the nucleus with
a cap, poly A tail, and no introns

• Exit through nuclear pore complexes
- Specific proteins called mRNPs associate with
processed mRNA molecules and direct them to
and through the nuclear pore
- mRNPs interact with a pore complexes
called the mRNA exporter
- Other proteins are involved

• mRNAs that fail to be spliced (introns removed) will
not exit the nucleus
- Why is this important? Protein will not be made

Transcription - Recapitulation
• All genes (eukaryotic and prokaryotic) have a unique region (promoters) of DNA sequence
upstream of (before) the transcriptional start site that serves as a binding site for the RNA
polymerase enzyme
• General functions of promoters:
1. Provides specificity – Each gene has a unique promoter sequence
2. Tell RNA polymerase where to start (they are usually at the beginning)
- No promoter (or mutated promoter), no transcription
3. Indicate which strand will be transcribed and the direction of transcription

• Most promoters share a few common sequences called consensus sequences
Prokaryotic promoters share 2 main types of consensus sequences
- TATA box (aka Pribnow box) (-10-15), sequence is usually TATAAT
- TTGACA – found 35 nucleotides upstream
• RNA polymerase enzyme makes direct contact with the promoter at these consensus
sequences
• Unique seq. within and around promoters help determine rates of transcription
Prokaryotic cells only produce 1 type of RNA polymerase to produce all of the mRNA, tRNA,
and rRNA in the cell.
- 2 alpha (α), 1 beta (β), 1 beta prime (β'), and 1 sigma (σ) subunit
- Different sigma factors recognize different promoter sequences.


- β, β’, α subunits interact and form the CORE ENZYME
- Different sigma factors recognize different promoter sequences
• Once it binds to the specific promoter, RNA polymerase positions itself over the
transcriptional start site based on its distance from the consensus sequences
- No specific “start signal” exists
• RNA pol unwinds the DNA near the start site (transcription bubble created).
• RNA pol continues producing a complimentary RNA molecule until it transcribes
a terminator signal.
Two major prokaryotic transcriptional terminator signals exist
- Both must: (a) Slow down the RNA polymerase; (b) Weaken the interaction between the DNA
and RNA in the bubble.
Properties of the two major prokaryotic terminator signals
1. Rho-independent terminators - The DNA sequence contains an inverted repeat followed
by a string of 6 adenines. Following their transcription, the inverted repeats form Hydrogen
bonds with each other within the RNA and form a hairpin loop
- Loop formation causes the RNA pol to pause
- The pause combined with weak hydrogen bonding between
6 straight A-U pairs cause the RNA to totally fall off of the
DNA template


• (… two major prokaryotic terminator signals)
2. Rho-dependent terminators - Also contain inverted repeats that cause hairpin
loop formation in the new RNA. Slows down the polymerase
- RNA contains a binding site for the Rho protein. Rho moves towards the 3’ end
- Acts as a RNA/DNA helicase. Rho is ATPase and helicase.
- Many prokaryotic genes are organized in operons (genes in tandem, in similar pathway).

Eukaryotic transcription follows the same basic steps of prokaryotic transcription
- Activation, initiation, elongation, termination
- Major differences exist in how they accomplish the above steps
• General features of transcriptional activation is similar in prokaryotic and eukaryotic cells
- Some genes are constituitivelly expressed, others are regulated
- The exact nature of the signals that activate transcription can be very different
• Initiation – Making the DNA accessible. Eukaryotic DNA is tightly coiled around proteins
called histones (and some nonhistone proteins)
- DNA is negatively charged (phosphate groups), histones are very positively charged
- A histone octamer (8 histone proteins in a complex). Each group is called a nucleosome
• DNA is freed from histones in 2 major ways:
1) Histone acetylation (HATs) add an acetyl group (CH3CO) to histones. This neutralizes their
positive charge and causes them to lose their attraction for DNA.
- Occurs only in the area to be transcribed (it is specfic!)
 Deacetylases remove acetyl groups after transcription
2) Chromatin remodeling - Some proteins move nucleosomes around without directly
modifying histones.

Lecture 6 (biol3600) transcription m rna processing- winter 2012 pw

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