Transcript Brooker Chapter 12 - Volunteer State Community College

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Genetics: Analysis and Principles
Robert J. Brooker
CHAPTER 12
GENE TRANSCRIPTION
AND
RNA MODIFICATION
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
Transcription literally means the act or
process of making a copy

DNA sequence to RNA sequence

1. DNA sequences provide the underlying
information


Signals for the start and end of transcription
2. Proteins recognize these sequences and
carry out the process

Other proteins modify the RNA transcript to make it
functionally active
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12-2
Gene Expression Requires
Base Sequences

At the molecular level, a gene is a transcriptional
unit

During gene expression, different types of base
sequences perform different roles

Figure 12.1 shows a common organization of
sequences within a bacterial gene and its transcript
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12-4

Bacterial transcriptional unit
• Start codon: specifies the first amino acid in a
protein sequence, usually a formylmethionine
(in bacteria) or a methionine (in eukaryotes)
Signals the end of
protein synthesis
• Bacterial mRNA may be polycistronic, which
means it encodes two or more polypeptides
Figure 12.1
12-5
A eukaryotic gene and its transcript
Gene Expression Requires
Base Sequences

The strand that is actually transcribed 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
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12-6
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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12-7
Initiation
The promoter functions as a recognition
site for transcription factors
The transcription factors enable RNA
polymerase to bind to the promoter
forming a closed promoter complex
Following binding, the DNA is denatured
into a bubble known as the open promoter
complex, or simply an open complex



Elongation
RNA polymerase slides along the DNA in
an open complex to synthesize the RNA
transcript

Termination

Figure 12.2
A termination signal is reached that
causes RNA polymerase to dissociated
from the DNA
12-8
The initiation of transcription at a eukaryotic promoter
RNA Transcripts Have Different
Functions

A structural gene is a one that encodes a
polypeptide


When such genes are transcribed, the product is an RNA
transcript called messenger RNA (mRNA)
Other RNA transcripts becomes part of a complex
that contains protein subunits

For example
 Ribosomes
 Spliceosomes
 Signal recognition particles
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12-10
12-11
12.2 TRANSCRIPTION IN
BACTERIA
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12-12
Promoters

Promoters are DNA sequences that “promote” gene
expression



Rate
Start site
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 12.3
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12-13
Sequence elements that play
a key role in transcription
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
Sometimes termed the
Pribnow box, after its
discoverer
Figure 12.3 The conventional numbering system of promoters
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12-14
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 12.4 Examples of –35 and –10 sequences within a variety of
bacterial promoters
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12-15
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


Four subunits = a2bb’
One subunit = s
These subunits play distinct functional roles
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12-16
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
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12-17
Amino acids within the
a helices hydrogen
bond with bases in the
promoter sequence
elements
Figure 12.5
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12-18

The binding of the RNA polymerase to the promoter
forms the closed complex

Then, the open complex is formed when the
TATAAT box 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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12-19
Figure 12.6
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12-20
Elongation in Bacterial Transcription

The RNA transcript is synthesized during the
elongation step

The DNA strand used as a template for RNA
synthesis is termed the template or noncoding strand

The opposite DNA strand is called the coding strand

It has the same base sequence as the RNA transcript

Except that T in DNA corresponds to U in RNA
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12-21
Similar to the
synthesis of DNA
via DNA polymerase
Figure 12.7
12-23
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
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12-24
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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12-29
Fig. 12.10a(TE Art)
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Structure of a bacterial
RNA polymerase
Structure of a eukaryotic
RNA polymerase II
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

Transcriptional start site
TATA box
Regulatory elements

Refer to Figure 12.11


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12-31
Figure 12.11

The core promoter is relatively short
 It consists of the TATA box


Usually an
adenine
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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12-32
Figure 12.11

Usually an
adenine
Regulatory elements affect the binding of RNA polymerase
to the promoter
 They are of two types
 Enhancers


Silencers


Stimulate transcription
Inhibit transcription
They vary in their locations but are often found in the
–50 to –100 region
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12-33
Sequences of Eukaryotic
Structural Genes

cis-acting elements



DNA sequences that exert their effect only on
nearby genes
Example: TATA box, enhancers and silencers
trans-acting elements

Regulatory proteins that bind to such DNA
sequences

Transcription factors
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12-34
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 12.12 shows the assembly of transcription
factors and RNA polymerase II at the TATA box
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12-35
Figure 12.12
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12-36
A closed complex
Figure 12.12

TFIIH plays a major role in the formation
of the open complex

It has several subunits that
perform different functions


One subunit hydrolyzes ATP and phosphorylates a
domain in RNA pol II known as the carboxyl terminal
domain (CTD)

This releases the contact between TFIIB and
RNA pol II
Other subunits act as helicases

Promote the formation of the open complex
RNA pol II can now
proceed to the
elongation stage
Released after the
open complex is
formed
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12-37

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
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12-38
12-39
Chromatin Structure and
Transcription

The compaction of DNA to form chromatin can be
an obstacle to the transcription pocess

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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12-40
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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12-41

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 12.13
Removes acetyl groups,
thereby restoring a
tighter interaction
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12-42

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
Figure 12.13
These effects may significantly alter
gene expression
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12-43
12.4 RNA MODIFICATION

Analysis of bacterial genes in the 1960s and 1970s
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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12-44
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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12-45
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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12-46
12-47
A eukaryotic gene and its transcript
Trimming

Many nonstructural genes are initially transcribed
as a large RNA

This large RNA transcript is enzymatically cleaved
into smaller functional pieces

Figure 12.14 shows the processing of mammalian
ribosomal RNA
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12-48
This processing occurs
in the nucleolus
Functional RNAs that are key
in ribosome structure
Figure 12.14
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12-49
Endonuclease
(Endonuclease)
RNase P
(RNase D)
Found to contain both RNA
and protein subunits
However, RNA contains the
catalytic ability
Covalently
modified bases
Therefore, it is a ribozyme
Figure 12.15
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12-51
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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12-52
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 12.16
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12-53
RNA displacement
loop
mRNA cannot hybridize to this
region
Because the intron has been
spliced out from the mRNA
Figure 12.16
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12-54
The Hypothesis

The b-globin gene from the mouse contains one
or more introns
Testing the Hypothesis

Refer to Figure 12.17
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12-55
Figure 12.17
12-56
The Data
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12-57
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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12-58
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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12-59

Splicing among group I and II introns is termed
self-splicing



Splicing does not require the aid of enzymes
Instead the RNA itself functions as its own ribozyme
Group I and II self-splicing can occur in vitro
without the additional proteins
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12-60
Figure 12.18
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12-61

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 12.16
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12-62

Table 12.4 describes the occurrence of introns in
genes of different species
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12-63
Capping

Most mature mRNAs have a 7-methyl guanosine
covalently attached at their 5’ end


This event is known as capping
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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12-64
Figure 12.19
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12-65
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 12.20
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12-68
Figure 12.20
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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12-69
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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12-70
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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12-71

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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12-72
Intron loops out and
exons brought closer
together
Figure 12.22
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12-73
Intron will be degraded and
the snRNPs used again
Figure 12.22
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12-74
Alternative RNA splicing
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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12-76