PowerPoint Presentation Materials
to accompany
Genetics: Analysis and Principles
Robert J. Brooker
CHAPTER 12
GENE TRANSCRIPTION AND
RNA MODIFICATION
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INTRODUCTION

At the molecular level, a gene is a segment
of DNA used to make a functional product


either an RNA or a polypeptide
Transcription is the first step in gene
expression
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12-2
Gene Expression

Structural genes encode the amino acids of a
polypeptide




Transcription of a structural gene produces messenger
RNA, usually called mRNA
The mRNA sequence determines the amino acids in the
polypeptide
The function of the protein determines traits
This path from gene to trait is called the central
dogma of genetics

Refer to Figure 12.1
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12-4
The central dogma of genetics
Figure 12.1
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12.1 OVERVIEW OF
TRANSCRIPTION

Gene expression is the overall process by which
the information within a gene is used to produce a
functional product which can determine a trait in
play with the environment

Figure 12.2 shows common organization of a
gene
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12-6
common organization of a gene
Signals the end of
protein synthesis
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Gene Expression Requires
Base Sequences


The strand that is actually transcribed (used as the
template) is termed the template strand
The opposite strand is called the coding strand or
the sense strand

The base sequence is identical to the RNA transcript



Except for the substitution of uracil in RNA for thymine in DNA
Transcription factors recognize the promoter and
regulatory sequences to control transcription
mRNA sequences such as the ribosomal-binding
site and codons direct translation
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12-8
The Stages of Transcription

Transcription occurs in three stages




Initiation
Elongation
Termination
These steps involve protein-DNA interactions

Proteins such as RNA polymerase interact with DNA
sequences
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12-9
Figure 12.3
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RNA Transcripts Have Different
Functions

The RNA transcripts from nonstructural genes are
not translated



They do have various important cellular functions
They can still confer traits
In some cases, the RNA transcript becomes part of a
complex that contains protein subunits

For example
 Ribosomes
 Spliceosomes
 Signal recognition particles
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12-12
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12.2 TRANSCRIPTION IN
BACTERIA
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12-14
Promoters

Promoters are DNA sequences that “promote” gene
expression


More precisely, they direct the exact location for the
initiation of transcription
Promoters are typically located just upstream of the
site where transcription of a gene actually begins

The bases in a promoter sequence are numbered in
relation to the transcription start site

Refer to Figure 12.4
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12-15
Most of the promoter region is
labeled with negative numbers
Bases preceding
this are numbered
in a negative
direction
There is no base
numbered 0
Bases to the right are
numbered in a
positive direction
Figure 12.4 The conventional numbering system of promoters
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12-16
Sequence elements that play
a key role in transcription
The promoter may span a large
region, but specific short
sequence elements are
particularly critical for promoter
recognition and activity level
Sometimes termed the
Pribnow box, after its
discoverer
Figure 12.4 The conventional numbering system of promoters
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12-17
For many bacterial
genes, there is a good
correlation between
the rate of RNA
transcription and the
degree of agreement
with the consensus
sequences
The most commonly
occurring bases
Figure 12.5 Examples of –35 and –10 sequences within a variety of
bacterial promoters
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12-18
Initiation of Bacterial Transcription

RNA polymerase is the enzyme that catalyzes the
synthesis of RNA

In E. coli, the RNA polymerase holoenzyme is
composed of

Core enzyme


Sigma factor


Five subunits = a2bb’
One subunit = s
These subunits play distinct functional roles
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12-19
Initiation of Bacterial Transcription

The RNA polymerase holoenzyme binds loosely to
the DNA

It then scans along the DNA, until it encounters a
promoter region

When it does, the sigma factor recognizes both the –35
and –10 regions


A region within the sigma factor that contains a helix-turn-helix
structure is involved in a tighter binding to the DNA
Refer to Figure 12.6
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12-20
Amino acids within the
a helices hydrogen
bond with bases in the
promoter sequence
elements
Figure 12.6
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12-21

The binding of the RNA polymerase to the promoter
forms the closed complex

Then, the open complex is formed when the
TATAAT box in the -10 region is unwound

A short RNA strand is made within the open
complex

The sigma factor is released at this point


This marks the end of initiation
The core enzyme now slides down the DNA to
synthesize an RNA strand
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12-22
Figure 12.7
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Elongation in Bacterial Transcription

The open complex formed by the action of RNA
polymerase is about 17 bases long

Behind the open complex, the DNA rewinds back into the
double helix

On average, the rate of RNA synthesis is about 43
nucleotides per second!

Figure 12.8 depicts the key points in the synthesis of
the RNA transcript
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12-25
Similar to the
synthesis of DNA
via DNA polymerase
Figure 12.8
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Termination of Bacterial
Transcription

Termination is the end of RNA synthesis

It occurs when the short RNA-DNA hybrid of the open
complex is forced to separate


This releases the newly made RNA as well as the RNA polymerase
E. coli has two different mechanisms for termination

1. rho-dependent termination


Requires a protein known as r (rho)
2. rho-independent termination

Does not require r
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12-27
rho utilization site
Rho protein is
a helicase
Figure 12.10 r-dependent termination
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Figure 12.10 r-dependent termination
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
r-independent termination is facilitated by two sequences in the RNA

1. A uracil-rich sequence located at the 3’ end of the RNA

2. A stem-loop structure upstream of the Us
URNA-ADNA hydrogen
bonds are very weak
Stabilizes
the RNA pol
pausing
No protein is required to physically
remove the RNA from the DNA
This type of termination
is also called intrinsic
Figure 12.11 r-independent termination
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12.3 TRANSCRIPTION IN
EUKARYOTES

Many of the basic features of gene transcription
are very similar in bacteria and eukaryotes

However, gene transcription in eukaryotes is more
complex


Larger organisms and cells
Cellular complexity such as organelles


added complexity means more genes
Multicellularity

increased regulation to express only in right cells at right time
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12-31
Eukaryotic RNA Polymerases

Nuclear DNA is transcribed by three different RNA
polymerases

RNA pol I


Transcribes all rRNA genes (except for the 5S rRNA)
RNA pol II

Transcribes all structural genes



Thus, synthesizes all mRNAs
Transcribes some snRNA genes
RNA pol III


Transcribes all tRNA genes
And the 5S rRNA gene
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12-32
Eukaryotic RNA Polymerases

All three are very similar structurally and are
composed of many subunits

There is also a remarkable similarity between the
bacterial RNA pol and its eukaryotic counterparts

Refer to Figure 12.12
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12-33
Sequences of Eukaryotic
Structural Genes

Eukaryotic promoter sequences are more variable
and often more complex than those of bacteria

For structural genes, at least three features are
found in most promoters

Regulatory elements
TATA box
Transcriptional start site

Refer to Figure 12.13


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12-34
Usually an
adenine

The core promoter is relatively short
 It consists of the TATA box


Important in determining the precise start point for transcription
The core promoter by itself produces a low level of
transcription

This is termed basal transcription
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12-35
Figure 12.13

Regulatory elements affect the binding of RNA polymerase
to the promoter
 They are of two types
 Enhancers


Silencers


Stimulate transcription
Inhibit transcription
They vary widely in their locations but are often found in
the –50 to –100 region
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12-36
Sequences of Eukaryotic
Structural Genes

Factors that control gene expression can be divided
into two types, based on their “location”

cis-acting elements



DNA sequences that exert their effect only over a
particular gene
Example: TATA box, enhancers and silencers
trans-acting elements

Regulatory proteins that bind to such DNA sequences
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12-37
RNA Polymerase II and its
Transcription Factors

Three categories of proteins are required for basal
transcription to occur at the promoter




RNA polymerase II
Five different proteins called general transcription factors
(GTFs)
A protein complex called mediator
Figure 12.14 shows the assembly of transcription
factors and RNA polymerase II at the TATA box
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A closed complex
Figure 12.14
CTD carboxy
terminal domain (CTD)
RNA pol II can now
proceed to the
elongation stage
Released after the
open complex is
formed
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12-40

Basal transcription apparatus


RNA pol II + the five GTFs
The third component for transcription is a large
protein complex termed mediator

It mediates interactions between RNA pol II and various
regulatory transcription factors

Its subunit composition is complex and variable

Mediator appears to regulate the ability of TFIIH to
phosphorylate CTD

Therefore it plays a pivotal role in the switch between
transcriptional initiation and elongation
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Chromatin Structure and
Transcription

The compaction of DNA to form chromatin can be
an obstacle to the transcription process

Most transcription occurs in interphase

Then, chromatin is found in 30 nm fibers that are
organized into radial loop domains

Within the 30 nm fibers, the DNA is wound around histone
octamers to form nucleosomes
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12-43
Chromatin Structure and
Transcription

The histone octamer is roughly five times smaller
than the complex of RNA pol II and the GTFs

The tight wrapping of DNA within the nucleosome
inhibits the function of RNA pol

To circumvent this problem, the chromatin structure
is significantly loosened during transcription

Two common mechanisms alter chromatin structure
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12-44

1. Covalent modification of histones

Amino terminals of histones are modified in various ways

Acetylation; phosphorylation; methylation
Adds acetyl groups, thereby
loosening the interaction
between histones and DNA
Figure 12.15
Removes acetyl groups,
thereby restoring a
tighter interaction
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12-45

2. ATP-dependent chromatin remodeling

The energy of ATP is used to alter the structure of
nucleosomes and thus make the DNA more accessible
Proteins are members of the
SWI/SNF family
Acronyms refer to the effects on yeast
when these enzyme are defective
Mutants in SWI are defective in
mating type switching
Mutants in SNF are
sucrose non-fermenters
These effects may significantly alter
gene expression
Figure 12.15
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12-46
12.4 RNA MODIFICATION

Analysis of bacterial genes in the 1960s and 1970
revealed the following:


The sequence of DNA in the coding strand corresponds to
the sequence of nucleotides in the mRNA
This in turn corresponds to the sequence of amino acid in
the polypeptide

This is termed the colinearity of gene expression

Analysis of eukaryotic structural genes in the late
1970s revealed that they are not always colinear
with their functional mRNAs
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12-47
12.4 RNA MODIFICATION

Instead, coding sequences, called exons, are
interrupted by intervening sequences or introns

Transcription produces the entire gene product



Introns are later removed or excised
Exons are connected together or spliced
This phenomenon is termed RNA splicing


It is a common genetic phenomenon in eukaryotes
Occurs occasionally in bacteria as well
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12-48
12.4 RNA MODIFICATION

Aside from splicing, RNA transcripts can be modified
in several ways
 For example



Trimming of rRNA and tRNA transcripts
5’ Capping and 3’ polyA tailing of mRNA transcripts
Refer to Table 12.3
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Processing

Many nonstructural genes are initially transcribed
as a large RNA

This large RNA transcript is enzymatically cleaved
into smaller functional pieces

Figure 12.16 shows the processing of mammalian
ribosomal RNA
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12-51
This processing occurs
in the nucleolus
Functional RNAs that are key
in ribosome structure
Figure 12.16
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12-52
Processing

Transfer RNAs are also made as large precursors

These have to be cleaved at both the 5’ and 3’ ends to
produce mature, functional tRNAs

This event has been studied extensively in E. coli

Figure 12.17 shows the trimming of a precursor
tRNA that carries the amino acid tyrosine (tRNAtyr)

Interestingly, the cleavage occurs differently at the 5’ end
and the 3’ end
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12-53
Found to contain both RNA
and protein subunits
However, RNA contains the
catalytic ability
Covalently
modified bases
Therefore, it is a ribozyme
Figure 12.17
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12-54
Experiment 12A: Identification of
Introns Via Microscopy

In the late 1970s, several research groups
investigated the presence of introns in eukaryotic
structural genes

One of these groups was led by Phillip Leder

Leder used electron microscopy to identify introns in
the b-globin gene


It had been cloned earlier
Leder used a strategy that involved hybridization
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12-55
Experiment 12A: Identification of
Introns Via Microscopy

Double-stranded DNA of the cloned b-globin gene
is first denatured


Then mixed with mature b-globin mRNA
The mRNA is complementary to the template
strand of the DNA

So the two will bind or hybridize to each other


If the DNA is allowed to renature, this complex will prevent the
reformation of the DNA double helix
Refer to Figure 12.18
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12-56
RNA displacement
loop
mRNA cannot hybridize to this
region
Because the intron has been
spliced out from the mRNA
Figure 12.18
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12-57
The Hypothesis

The b-globin gene from the mouse contains one
or more introns
Testing the Hypothesis

Refer to Figure 12.19
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12-58
Figure 12.19
12-59
The Data
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12-60
Interpreting the Data
Hybridization caused the
formation of two R loops,
separated by a doublestranded DNA region
This suggests that the b-globin
gene contains introns
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12-61
Splicing

Three different splicing mechanisms have been
identified




Group I intron splicing
Group II intron splicing
Spliceosome
All three cases involve


Removal of the intron RNA
Linkage of the exon RNA by a phosphodiester bond
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12-62

Splicing among group I and II introns is termed
self-splicing



Group I and II differ in the way that the intron is
removed and the exons reconnected


Splicing does not require the aid of enzymes
Instead the RNA itself functions as its own ribozyme
Refer to Figure 12.20
Group I and II self-splicing can occur in vitro
without the additional proteins

However, in vivo, proteins known as maturases often
enhance the rate of splicing
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12-63
Figure 12.20
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12-64

In eukaryotes, the transcription
of structural genes, produces a
long transcript known as
pre-mRNA

Also as heterogeneous nuclear
RNA (hnRNA)

This RNA is altered by splicing
and other modifications, before
it leaves the nucleus

Splicing in this case requires
the aid of a multicomponent
structure known as the
spliceosome
Figure 12.20
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12-65
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12-66
Pre-mRNA Splicing

The spliceosome is a large complex that splices
pre-mRNA

It is composed of several subunits known as
snRNPs (pronounced “snurps”)

Each snRNP contains small nuclear RNA and a set of
proteins
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12-67
Pre-mRNA Splicing

The subunits of a spliceosome carry out several
functions

1. Bind to an intron sequence and precisely recognize
the intron-exon boundaries

2. Hold the pre-mRNA in the correct configuration

3. Catalyze the chemical reactions that remove introns
and covalently link exons
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12-68

Intron RNA is defined by particular sequences within the
intron and at the intro-exon boundaries

The consensus sequences for the splicing of mammalian
pre-mRNA are shown in Figure 12.21
Sequences shown in bold
are highly conserved
Figure 12.21

Corresponds to the boxed
adenine in Figure 12.22
Serve as recognition sites for the
binding of the spliceosome
The pre-mRNA splicing mechanism is shown in Figure 12.22
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12-69
Intron loops out and
exons brought closer
together
Figure 12.22
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12-70
Intron will be degraded and
the snRNPs used again
Figure 12.22
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12-71
Intron Advantage?

One benefit of genes with introns is a phenomenon
called alternative splicing

A pre-mRNA with multiple introns can be spliced in
different ways


This will generate mature mRNAs with different
combinations of exons
This variation in splicing can occur in different cell
types or during different stages of development
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12-72
Intron Advantage?

The biological advantage of alternative splicing is
that two (or more) polypeptides can be derived
from a single gene

This allows an organism to carry fewer genes in its
genome
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12-73
Capping

Most mature mRNAs have a 7-methyl guanosine
covalently attached at their 5’ end


Capping occurs as the pre-mRNA is being
synthesized by RNA pol II


This event is known as capping
Usually when the transcript is only 20 to 25 bases long
As shown in Figure 12.23, capping is a three-step
process
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12-74
Figure 12.23
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Figure 12.23
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12-76
Capping

The 7-methylguanosine cap structure is recognized
by cap-binding proteins

Cap-binding proteins play roles in the



Movement of some RNAs into the cytoplasm
Early stages of translation
Splicing of introns
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12-77
Tailing

Most mature mRNAs have a string of adenine
nucleotides at their 3’ ends


The polyA tail is not encoded in the gene sequence


This is termed the polyA tail
It is added enzymatically after the gene is completely
transcribed
The attachment of the polyA tail is shown in
Figure 12.24
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12-78
Figure 12.24
Consensus sequence in
higher eukaryotes
Appears to be important in the
stability of mRNA and the
translation of the polypeptide
Length varies between species
From a few dozen adenines
to several hundred
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12-79