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INTRODUCTION

• At the molecular level, a gene is a segment

of DNA used to make a functional product
Chapter 12
– either an RNA or a polypeptide
Lecture Outline
See separate PowerPoint slides for all figures and tables pre-
inserted into PowerPoint without notes.
• Transcription is the first step in gene
expression

Copyright © McGraw-Hill Education. Permission required for reproduction or display. 1 12-2

TRANSCRIPTION Gene Expression

 Structural genes or Protein-encoding genes encode
the amino acid sequence of a polypeptide
• Transcription literally means the act or process of
 Transcription of a structural gene produces messenger
making a copy RNA, usually called mRNA
 The mRNA nucleotide sequence determines the amino
• In genetics, the term refers to the copying of a acid sequence of a polypeptide during translation
DNA sequence into an RNA sequence  The synthesis of functional proteins determines an
organisms traits

• The structure of DNA is not altered as a result of

this process  This path from gene to trait is called the central
– It can continue to store information
dogma of genetics
 Refer to Figure 12.1

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The central dogma of genetics 12.1 OVERVIEW OF

TRANSCRIPTION
• A key concept is that DNA base sequences define the
beginning and end of a gene and regulate the level of
RNA synthesis

• Another important concept is that proteins must

recognize and act on DNA for transcription to occur

• Gene expression is the overall process by which the

information within a gene is used to produce a
functional product which can, in concert with
environmental factors, determine a trait

• Figure 12.2 shows common organization of a

bacterial gene
Figure 12.1
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Gene Expression Requires Base

Sequences
 The DNA strand that is actually transcribed (used
as the template) is termed the template strand
 The RNA transcript is complementary to the template strand
 The opposite strand is called the coding strand or
Signals the start of
RNA synthesis
Signals the end of
RNA synthesis
the sense strand as well as the nontemplate 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
Specifies the start
of protein synthesis  mRNA sequences such as the ribosomal-binding
Specifies the end of
protein synthesis site and codons direct translation
Figure 12.2
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The Stages of Transcription Transcription

 Transcription occurs in three stages

 Initiation
 Elongation
 Termination

 These steps involve protein-DNA interactions

 Proteins such as RNA polymerase interact with DNA
sequences

Figure 12.3
12-9 12-10

RNA Transcripts Have Different RNA Transcripts Have Different

Functions Functions
 Once they are made, RNA transcripts play different  The RNA transcripts from nonstructural genes are
functional roles not translated
 Refer to Table 12.1
 They do have various important cellular functions
 Well over 90% of all genes are structural genes  They can still confer traits
which are transcribed into mRNA  In some cases, the RNA transcript becomes part of a
 Final functional products are polypeptides complex that contains protein subunits
 The other RNA molecules in Table 12.1 are never  For example
 Ribosomes
translated
 Spliceosomes
 Final functional products are RNA molecules  Signal recognition particles

 Telomerase

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12.2 TRANSCRIPTION IN
BACTERIA
• Our molecular understanding of gene transcription
came from studies involving bacteria and
bacteriophages

• Indeed, much of our knowledge comes from

studies of a single bacterium
– E. coli, of course

• In this section we will examine the three steps of

transcription as they occur in bacteria

12-13 12-14

Bases preceding the

Most of the promoter region is start site are
labeled with negative numbers numbered in a

Promoters negative direction

There is no base
numbered 0
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Coding strand Transcriptional

Promoter region start site
 Promoters are DNA sequences that “promote” gene
expression –35 sequence 16 –18 bp –10 sequence
+1
 More precisely, they direct the exact location for the 5′ 3′
initiation of transcription TT C
GA A
T T
A AA
T
A
AA G A A A
 Promoters are typically located just upstream of the CT T T T T T
site where transcription of a gene actually begins 3′ 5′

 The bases in a promoter sequence are numbered in Template strand 5′ 3′

relation to the transcription start site Bases to the right are
numbered in a RNA
positive direction
A

 Refer to Figure 12.4 Transcription

Figure 12.4 The conventional numbering system of promoters

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The promoter may span a large

Sequence elements that play
region, but specific short
a key role in transcription
sequence elements are
particularly critical for promoter
recognition and activity level

Coding strand Transcriptional For many bacterial

genes, there is a good
Promoter region start site correlation between
the rate of RNA
–35 sequence 16 –18 bp –10 sequence transcription and the
degree of agreement
+1 with the consensus
5′ 3′ sequences of the -35
and -10 regions
TT C T T T
GA A A AA A
AA G A A A
CT T T T T T
3′ 5′

Template strand Sometimes termed the

5′ 3′ The most commonly
Pribnow box, after its occurring bases
discoverer RNA
A
Transcription
Figure 12.4 The conventional numbering system of promoters
Figure 12.5 Examples of –35 and –10 sequences within a variety of bacterial promoters
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Initiation of Bacterial Transcription Initiation of Bacterial Transcription

 RNA polymerase is the enzyme that catalyzes the  The RNA polymerase holoenzyme binds loosely to
synthesis of RNA the DNA

 In E. coli, the RNA polymerase holoenzyme is  It then scans along the DNA, until it encounters a
composed of promoter region
 Core enzyme  When it does, the sigma factor recognizes both the –35
 Five subunits = a2bb’ and –10 regions
 Sigma factor  A region within the sigma factor that contains a helix-turn-helix
 One subunit = s structure is involved in a tighter binding to the DNA

 These subunits play distinct functional roles  Refer to Figure 12.6

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The binding of the RNA polymerase to the promoter

Binding of sfactor protein to DNA double helix 
forms the closed complex

 Then, the open complex is formed when the

TATAAT box in the -10 region is unwound
 A-T bonds are more easily separated
Amino acids within the  A short RNA strand is made within the open
a helices hydrogen
bond with bases in the complex
-35 and -10 promoter
sequences  The sigma factor is released at this point
 This marks the end of initiation
 See Figure 12.7
 The core enzyme now slides down the DNA to
synthesize an RNA strand
 This is known as the elongation phase
Figure 12.6
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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
RNA polymerase

σ factor
Promoter region
–35 –10 Elongation in Bacterial Transcription
After sliding along the DNA, σ
factor recognizes a promoter, and
RNA polymerase
RNA polymerase holoenzyme
holoenzyme
forms a closed complex.
–35  The RNA transcript is synthesized during the
–10
elongation stage
Closed complex

An open complex is formed, and

a short RNA is made.  The DNA strand used as a template for RNA
–35
–10 synthesis is termed the template or antisense strand
Open complex

σ factor is released, and the

core enzyme is able to proceed
 The opposite DNA strand is called the coding strand
down the DNA.
–35
–10
RNA polymerase
core enzyme  It has the same base sequence as the RNA transcript
 Except that T in DNA corresponds to U in RNA
σ factor

RNA transcript
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 a
double helix

 On average, the rate of RNA synthesis is about 43

nucleotides per second!
Similar to the
synthesis of DNA
via DNA polymerase
 Figure 12.8 depicts the key points in the synthesis of
an RNA transcript
Figure 12.8
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Both strands of genomic DNA are used as template Termination of Bacterial

strand for different genes
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
Transcription on  Does not require r
opposite strand, but
still in 5’ to 3’ direction
Figure 12.9
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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
rho utilization
site
– 2. A stem-loop structure upstream of the uracil-rich sequence

Rho protein is
a helicase

Stabilizes
URNA-ADNA hydrogen the RNA pol
bonds are relatively pausing
weak

No protein is required to physically

remove the RNA from the DNA
This type of termination
is also called intrinsic

Figure 12.10 r-dependent termination Figure 12.11 r-independent termination

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12.3 TRANSCRIPTION IN Eukaryotic RNA Polymerases

EUKARYOTES
• Many of the basic features of gene transcription  Nuclear DNA is transcribed by three different RNA
are very similar in bacteria and eukaryotes polymerases
 RNA pol I
 Transcribes all rRNA genes (except for the 5S rRNA)
• However, gene transcription in eukaryotes is more  RNA pol II
complex  Transcribes all protein-encoding (structural) genes
– Larger, more complex cells (organelles)  Thus, synthesizes all mRNAs
 Transcribes some snRNA genes needed for splicing
– Added cellular complexity means more genes that
encode proteins are required  RNA pol III
 Transcribes all tRNA genes
– Multicellularity adds another level of regulation
 And the 5S rRNA gene
• express genes only in the correct cells at the proper time
 microRNA genes
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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Eukaryotic RNA Polymerases

 All three are very similar structurally and are
composed of many subunits
Clamp controls
 There is also a remarkable similarity between the movement of DNA
Structure of through enzyme
bacterial RNA pol and its eukaryotic counterparts RNA polymerase Rudder forces RNA-
DNA hybrid apart

 Refer to Figure 12.12

RNA-DNA hybrid forced
to bend facilitating
addition of nucleotides
to template
DNA enters
through jaw
Figure 12.12
a(left): Courtesy of Dr. Seth Darst; a(right): From: Patrick Cramer, David A. Bushnell, Roger D. Kornberg (2001), “Structural Basis of
Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution,” Science, 292(5523):1863-1876, Fig. 1. Image courtesy of David A. Bushnell

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Figure 12.13
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Sequences of Eukaryotic Core promoter

TATA box
Transcriptional
start site
Usually an

Structural Genes Common location for

Coding-strand sequences:

regulatory elements such

TATAAA Py2CAPy5
adenine

–100 –50 –25 +1

as GC and CAAT boxes

 Eukaryotic promoter sequences are more variable DNA Transcription

and often more complex than those of bacteria

• The core promoter is relatively short
 Most eukaryotic genes have two features – It consists of the TATA box and transcriptional start site
• Important in determining the precise start point for transcription
 Core Promoter
 typically sequence TATAAA
• The core promoter by itself produces a low level of
 called TATA box
transcription
 transcriptional start site – This is termed basal transcription
 Regulatory elements
 Refer to Figure 12.13
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Figure 12.13
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Core promoter

TATA box
Transcriptional
start site
Sequences of Eukaryotic Structural
Common location for
Coding-strand sequences:

regulatory elements such

TATAAA Py2CAPy5
Genes
–100 –50 –25 +1
as GC and CAAT boxes

DNA Transcription  Factors that control gene expression can be divided

into two types, based on their “location”
• Regulatory elements are short DNA sequences that affect the
binding of RNA polymerase to the promoter
• Transcription factors (proteins) bind to these elements and  cis-acting elements
influence the rate of transcription
– They are two types of regulatory elements  DNA sequences that exert their effect only over a
• Enhancers particular gene
– Stimulate transcription  Example: TATA box, enhancers and silencers
• Silencers
– Inhibit transcription
 trans-acting elements
– They vary widely in their locations but are often found in the  Regulatory proteins that bind to such DNA sequences
–50 to –100 region

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Figure 12.14

RNA Polymerase II and its

Transcription Factors
 Three categories of proteins are required for basal
transcription to occur at the promoter
 RNA polymerase II
A closed complex
 Five different proteins called general transcription factors
(GTFs)
 A protein complex called mediator

RNA pol II can now

 Figure 12.14 shows the assembly of transcription Released after the
proceed to the
elongation stage
factors and RNA polymerase II at the TATA box open complex is
formed

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 Basal transcription apparatus

 RNA pol II + the five GTFs

 The third component required for transcription is a

large protein complex termed mediator
 It mediates interactions between RNA pol II and various
regulatory transcription factors that bind enhancers or
silencers
 Its subunit composition is complex and variable
 Core subunits partially wraps around RNA pol II
 Mediator may phosphorylate the CTD of RNA
polymerase II and it may regulate the ability of TFIIH to
phosphorylate the CTD
 Therefore it plays a pivotal role in the switch between
transcriptional initiation and elongation
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Possible mechanisms for Pol II termination

RNA Pol II transcriptional termination
• Pre-mRNAs are modified by cleavage near
their 3’ end with subsequent attachment of a
string of adenines
• Transcription terminates 500 to 2000
nucleotides downstream from the polyA signal
• There are two models for termination
– Further research is needed to determine if either,
or both are correct
• Refer to figure 12.15
Figure 12.15

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12.4 RNA MODIFICATION RNA Modification

• Analysis of bacterial genes in the 1960s and 1970 • Instead, coding sequences, called exons, are
revealed the following: interrupted by intervening sequences or introns
– The sequence of DNA in the coding strand corresponds to
the sequence of nucleotides in the mRNA
• Transcription produces a pre-mRNA corresponding
– The sequence of codons in the mRNA provides the
instructions for the sequence of amino acids in the to the entire gene sequence
polypeptide – Introns are later removed or excised
• This is termed the colinearity of gene expression – Exons are connected together or spliced

• Analysis of eukaryotic structural genes in the late • This phenomenon is termed RNA splicing
1970s revealed that they are not always colinear
with their functional mRNAs – It is a common genetic phenomenon in eukaryotes
– Occurs occasionally in bacteria as well

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RNA Modification
• Aside from splicing, RNA transcripts can be modified
in several ways
– For example
• Processing of rRNA and tRNA transcripts to smaller
functional pieces
• 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 This processing occurs
in the nucleolus

 Figure 12.16 shows the processing of mammalian

ribosomal RNA
Functional RNAs that are key
in ribosome structure

Figure 12.16
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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
 Cleaved by exonuclease and endonuclease
 exonucleases cleave a covalent bond between two nucleotides at
one end of a strand
Covalently
 endonucleases can cleave bonds within a strand modified bases

 Figure 12.17 shows the trimming of a precursor Found to contain both RNA
and protein subunits
tRNA However, RNA contains the
catalytic activity
 Interestingly, the cleavage occurs differently at the 5’ end
Therefore, it is a ribozyme
and the 3’ end
Figure 12.17
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Experiment 12A: Identification of Experiment 12A: Identification of

Introns Via Microscopy Introns Via Microscopy
• In the late 1970s, several research groups • Double-stranded DNA of the cloned b-globin gene
investigated the presence of introns in eukaryotic is first denatured
structural genes – Then mixed with mature b-globin mRNA

• One of these groups was led by Phillip Leder • The mRNA is complementary to the template
strand of the DNA
– Leder used electron microscopy to identify introns in
– So the two will bind or hybridize to each other
the b-globin gene
• If the DNA is allowed to renature, this complex will prevent the
• It had been cloned earlier
reformation of the DNA double helix
– Leder used a strategy that involved hybridization
– Refer to Figure 12.18

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RNA displacement
loop

The Hypothesis

– The b-globin gene from the mouse contains one

or more introns

mRNA cannot hybridize here

Because the intron has been
spliced out of the mRNA
Testing the Hypothesis
 Refer to Figure 12.19

Figure 12.18
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The Data Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Intron
R loop

R loop
aarssrraras-1978752727sasba

Data from: Tilghman, S. M., Tiemeier, D. C., Seidman, J. G., Peterlin, B. M.,
Sullivan, M., Maizel, J.V., and Leder, P. (1978) Intervening sequence of
DNA identified in the structural portion of a mouse beta-globin gene.
Figure 12.19 Proc. Natl. Acad. Sci. USA 75:725–729.
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Interpreting the Data

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Splicing
 Three different splicing mechanisms have been
identified
 Group I intron splicing
 Group II intron splicing
R loop
Intron  Spliceosome

 All three cases involve

Hybridization caused the
formation of two R loops, R loop  Removal of the intron RNA
separated by a double- a ar s    srra r  as -  1978 752727s a s  ba

stranded DNA region Data from: Tilghman, S. M., Tiemeier, D. C., Seidman, J. G., Peterlin, B. M.,
Sullivan, M., Maizel, J.V., and Leder, P. (1978) Intervening sequence of
 Linkage of the exon RNA by a phosphodiester bond
This suggests that the b-globin DNA identified in the structural portion of a mouse beta-globin gene.
gene contains introns Proc. Natl. Acad. Sci. USA 75:725–729.

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 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

2’ hydroxyl from
 Group I and II differ in the way that the intron is Free guanosine bound to adenine within
intron breaks bond
removed and the exons connected site in intron breaks the
bond between exon 1 and between exon 1 and
intron and becomes intron
 Refer to Figure 12.20 attached to 5’ end of
intron

Results in Exon 1 and 2 Results in Exon 1

 Group I and II self-splicing can occur in vitro covalently joined and free, and 2 linkage and
linear intron intron as lariat
without the additional proteins
 However, in vivo, proteins known as maturases often
enhance the rate of splicing
Figure 12.20
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 In eukaryotes, the transcription

of structural genes produces a
long transcript known as
pre-mRNA

 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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Pre-mRNA Splicing Pre-mRNA Splicing

 The spliceosome is a large complex that splices  The subunits of a spliceosome carry out several
pre-mRNA functions

 It is composed of several subunits known as  1. Bind to an intron sequence and precisely recognize
the intron-exon boundaries
snRNPs (pronounced “snurps”)
 Each snRNP contains small nuclear RNA and a set of
proteins  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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 Intron RNA is defined by particular sequences within the

intron and at the intron-exon boundaries

 The consensus sequences for the splicing of mammalian

pre-mRNA are shown in Figure 12.21
Corresponds to the boxed
Sequences shown in bold adenine in Figure 12.22
are highly conserved

Intron loops out and

exons brought closer
together

Figure 12.21 Serve as recognition sites for the

binding of the spliceosome

 The pre-mRNA splicing mechanism is shown in Figure 12.22

Figure 12.22
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Intron Advantage?
Cleavage may be
catalyzed by snRNA  One benefit of genes with introns is a phenomenon
molecules within U2
and U6 called alternative splicing

 A pre-mRNA with multiple introns can be spliced in

different ways
Intron will be degraded and
 This will generate mature mRNAs with different
the snRNPs used again combinations of exons

 This variation in splicing can occur in different cell

types or during different stages of development
Figure 12.22
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Intron Advantage? Capping

 The biological advantage of alternative splicing is  Most mature mRNAs have a 7-methylguanosine
that two (or more) polypeptides can be derived covalently attached at their 5’ end
from a single gene  This event is known as capping

 This allows an organism to carry fewer genes in its  Capping occurs as the pre-mRNA is being
genome synthesized by RNA pol II
 Usually when the transcript is only 20 to 25 bases long

 Molecular mechanism of alternative splicing will be  As shown in Figure 12.23, capping is a three-step
covered in Chapter 16 process

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5’ to 5’ triphosphate
linkage

Figure 12.23 Figure 12.23

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Capping Polyadenylation
 The 7-methylguanosine cap structure is recognized  Most mature mRNAs have a string of adenine
by cap-binding proteins nucleotides at their 3’ ends
 This is termed the polyA tail
 Cap-binding proteins play roles in the
 The polyA tail is not encoded in the gene sequence
 Movement of some RNAs into the cytoplasm  It is added enzymatically after the gene is completely
 Early stages of translation transcribed
 Splicing of introns
 The attachment of the polyA tail is shown in
Figure 12.24

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Figure 12.24 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Polyadenylation signal sequence

5′ AAUAAA 3′ RNA editing
Endonuclease cleavage occurs
Consensus sequence in
higher eukaryotes
about 20 nucleotides downstream  Change in the nucleotide sequence of an RNA
from the AAUAAA sequence.
 Can involve addition or deletion of particular bases
5′ AAUAAA  Can also occur through conversion of a base
PolyA-polymerase adds  First discovered in trypanosomes
adenine nucleotides  Now known to occur in many organisms
to the 3′ end.

5′ AAUAAA AAAAAAAAAAAA.... 3′  Refer to Table 12.5 for a list of examples

Appears to be important in PolyA tail
export from the nucleus, the
stability of mRNA and the
Length varies between mRNAs
translation of the polypeptide
Maximum length around 250
nucleotides

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RNA editing RNA editing

RNA editing can occur by changing one type
of base to another

Figure 12.25 Deamination converts RNA nucleotides to

new forms

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12.5 COMPARISON BETWEEN

BACTERIA AND EUKARYOTES

Table 12.6

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