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BIO 184
Fall 2006
LECTURE 2
Lecture 2:
Gene Transcription
The Central Paradigm of Molecular Genetics. Genes are transcribed into messenger RNA
molecules, which are then translated into polypeptides. The polypeptides then fold and may
combine with other polypeptides to from a function protein. This diagram is simplistic, however,
because genes can also be transcribed into small RNAs (tRNAs, rRNAs, etc.) that are never
translated and serve important functions in the cell as RNAs.
I. Overview of Transcription
Transcription literally means the act or process of making a copy
of something. Legal secretaries, for example, transcribe the taped
conversations between lawyers and clients by typing them into a
word-processing program.
Note, however, that transcription always maintains the original language of the
copied material. If the client and lawyer talk in Japanese, the transcript will also
be written in Japanese.
In genetics, “transcription” refers to the copying of a DNA sequence into an RNA
sequence
 The structure of DNA is not altered as a result of this process, and it
continues to store information.
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LECTURE 2
At the molecular level, a gene is a transcriptional unit
 Genes are defined as DNA sequences that are transcribed into RNA
The figure below shows a common organization of sequences within a bacterial
polypeptide-coding gene and its mRNA.
 Each gene has a promoter and one or more regulatory sequences.
o The promoter “promotes” transcription
o The regulatory sequences control when and where (in what cell type)
the gene will be expresssed
 The promoter and the regulatory sequences are DNA sequences that are not
part of the transcript. They are recognized and bound by DNA-binding
proteins.
• 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
The mRNA contains sequences that are recognized by the translation machinery
(the ribosome). The start and stop codons are not important during transcription
but are crucial signals during translation.
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BIO 184
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LECTURE 2
II. The Template and Coding Strands
RNA is fundamentally single-stranded and therefore only one strand of the DNA is
actually copied into RNA during transcription.
The strand that is actually being copied is termed the template strand.
 The RNA transcript will have the opposite polarity and the complementary
sequence to this strand
The opposite strand is called the coding strand
 The base sequence of this strand is identical in polarity and sequence to the
RNA transcript
o Except for the substitution of uracil in RNA for thymine in DNA
 Because the coding strand has the same sequence and polarity as the RNA, it
is said to “carry the gene.”
The gene is located on
the coding strand
TEMPLATE
CODING
III. The Stages of Transcription
See Figure 12.12, Brooker
IV. The Many Roles of RNA Transcripts
Once they are made, RNA transcripts play many different functional roles in the
cell:
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BIO 184
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LECTURE 2
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About 90% of the genes in most organisms code for mRNAs that are
ultimately translated into polypeptides.

However, tRNAs, rRNAs, and other RNAs that do not code for
polypeptides have very important roles in cellular processes.
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BIO 184
Fall 2006
LECTURE 2
V. Gene Transcription in Bacteria
Our molecular understanding of gene transcription came from studies involving
bacteria (mostly E. coli) and the viruses that infect them (bacteriophages).
A. 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” (5’) 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, which is labeled “+1”
The promoter attracts RNA polymerase, the enzyme responsible for transcribing
RNA, to the gene. Without a promoter, a gene sequence would not be transcribed.
Promoters often consist of conserved DNA sequences separated by a certain
distance that is conserved from one gene in the organism to the next.
 This is usually because the RNA polymerase enzyme binds to the DNA in one
spot and then arches over the DNA to bind to the next spot.
 If the distance between the recognition sites for the enzyme are shortened
or lengthened, transcription will not occur
o The RNA polymerase cannot fit onto the DNA anymore in its “lock and
key” position
See Figures 12.3, 12.4, Brooker
B. Initiation

In E. coli, the RNA polymerase holoenzyme is composed of
o Core enzyme
 Four subunits = a2bb’
o Sigma factor
 One subunit = s

These subunits play distinct functional roles
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Fall 2006
LECTURE 2
At the start of initiation, the RNA polymerase holoenzyme binds loosely to
the DNA
It then scans along the DNA, until it encounters a promoter
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
then interacts strongly with the promoter, causing RNA polymerase to
“tighten its grip” on the DNA.
Amino acids within the
 helices hydrogen
bond with bases in the
promoter sequence
elements

The tight binding of the RNA polymerase to the promoter forms what is
called the closed complex

Then, the open complex is formed when RNA polymerase denatures the
double-stranded DNA in the AT-rich Pribnow Box

Next, the RNA polymerase makes a short RNA strand copy of the template
strand within the denatured region
o The sigma factor is released at this point
o This marks the end of initiation
o Note that RNA polymerase, unlike DNA polymerase, is a “smart
enzyme”! It can start an RNA strand all on its own.
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LECTURE 2
The core enzyme now slides down the DNA to synthesize the transcript
C. Elongation
The RNA transcript is synthesized during the elongation step
 The open complex formed by the action of RNA polymerase is about 17
bases long and remains that size as the polymerase moves along the DNA
o Behind the open complex, the DNA rewinds back into the double helix
o
 On average, the rate of RNA synthesis is about 43 nucleotides per second
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LECTURE 2
Similar to the
synthesis of DNA
via DNA polymerase
D. Termination
Termination is the end of RNA synthesis
 It occurs when the short RNA-DNA hybrid of the open complex is forced to
separate
o This releases the newly made RNA as well as the RNA polymerase

E. coli has two different mechanisms for termination
o 1. rho-dependent termination
 Requires a protein known as r (rho)
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LECTURE 2
o 2. rho-independent termination
 Does not require r
See Figures 12.8, 12.9, Brooker
VI. Gene 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
o Larger organisms
o Cellular complexity
o Multicellularity
A. Eukaryotic RNA Polymerases
In eukaryotes, DNA is transcribed by three different RNA polymerases
 RNA pol I
o Transcribes all rRNA genes (except for the 5S rRNA)
 RNA pol II
o Transcribes all structural genes
 Thus, synthesizes all mRNAs
 Transcribes some snRNA genes
 RNA pol III
o Transcribes all tRNA genes
o And the 5S rRNA gene
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
B. Eukaryotic Polypeptide-Coding Genes
Eukaryotic promoter sequences are more variable and often more complex than
those of bacteria
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For genes coding for mRNAs, at least three features are found in most
promoters
o Transcriptional start site
o TATA box
o Regulatory elements

The core promoter is relatively short
o 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
o This is termed basal transcription

Regulatory elements affect the binding of RNA polymerase to the promoter
o They are of two types
 Enhancers
 Stimulate transcription
 Silencers
 Inhibit transcription
o They vary in their locations but are often found in the –50 to –100
region
C. CIS and TRANS Factors

cis-acting elements
o DNA sequences that exert their effect only on nearby genes
o Example: TATA box, enhancers and silencers

trans-acting elements
o Regulatory proteins that bind to such DNA sequences
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BIO 184
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LECTURE 2
D. Trans Factors Involved in Eukaryotic Transcription
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
The figure below shows the assembly of transcription factors and RNA polymerase
II at the TATA box
See Figure 12.12, Brooker
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LECTURE 2
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Basal transcription apparatus
o RNA pol II + the five GTFs

The third component for transcription is a large protein complex termed
mediator
o It mediates interactions between RNA pol II and various regulatory
transcription factors
o Its subunit composition is complex and variable
o 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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LECTURE 2
VII. RNA Processing in Eukaryotes
A. Splicing
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 genes in the late
1970s revealed that they are not always
colinear with their functional mRNAs
 Instead, coding sequences, called
exons, are interrupted by
intervening sequences or introns
 Transcription produces the entire
gene product
o Introns are later removed or
excised
o Exons are later spliced
together
This phenomenon is termed RNA splicing
 It is a common genetic phenomenon
in eukaryotes
 Occurs occasionally in bacteria as
well
The initial transcription produces a long
transcript known as a pre-mRNA

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Splicing requires the aid of a multicomponent structure known as the
spliceosome
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LECTURE 2
Why splice?
One benefit of genes with introns is a phenomenon called alternative splicing
 A pre-mRNA with multiple introns can be spliced in different ways
o 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
B. 5’ Capping
Most mature mRNAs have a 7-methyl guanosine covalently attached at their 5’ end
 This event is known as capping
Capping occurs as the pre-mRNA is being synthesized by RNA pol II
 Usually when the transcript is only 20 to 25 bases long
The “cap” consists of a backwards methylated guanine with a triphosphate link to
the 5’ nucleotide in the mRNA. It is added on and is not part of the original
transcript.
It is bound by cap binding proteins, which, in turn, are recognized and bound by
the ribosome during translation initiation.
Thus, the cap “marks” the RNA as an mRNA and aids in its recognition by the
ribosome for translation.
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BIO 184
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LECTURE 2
C. 3’ Polyadenylation
Most mature mRNAs have a string of adenine nucleotides at their 3’ ends
 This is termed the polyA tail
The polyA tail, like the 5’ cap, is not encoded in the gene sequence
 It is added enzymatically after the gene is completely transcribed
See Figure 12.20, Brooker
D. Example of pre-mRNA Processing
The beta-globin gene has 3 exons and 2 introns.
 The pre-mRNA (also called hnRNA) contains the entire gene sequence
starting at the +1 site and ending past the polyadenylation signal
 During processing, the introns are spliced out, the 5’ end of the mRNA is
capped, and the polyadenylation signal is cut and tailed.
From Principles of Genetics, 7th ed., R. H. Tamarin, McGraw Hill, 2002
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