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1. DNA sequences provide the underlying information
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Signals for the start and end of transcription
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2. Proteins recognize these sequences and carry out the process
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Other proteins modify the RNA transcript to make it functionally active
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At the molecular level, a gene is a transcriptional unit
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During gene expression, different types of base sequences perform different roles
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Figure 12.1 shows a common organization of sequences within a bacterial gene and its transcript
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Bacterial transcriptional unit
Figure 12.1
• 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
12-5

A eukaryotic gene and its transcript

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The strand that is actually transcribed is termed the template strand
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The opposite strand is called the coding strand or the sense strand
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The base sequence is identical to the RNA transcript
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Except for the substitution of uracil in RNA for thymine in DNA
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Transcription occurs in three stages
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Initiation
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Elongation
Termination
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These steps involve protein-DNA interactions
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Proteins such as RNA polymerase interact with DNA sequences
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Figure 12.2
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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
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Elongation
RNA polymerase slides along the DNA in an open complex to synthesize the RNA transcript
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Termination
A termination signal is reached that causes RNA polymerase to dissociated from the DNA
12-8

The initiation of transcription at a eukaryotic promoter

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A structural gene is a one that encodes a polypeptide
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When such genes are transcribed, the product is an RNA transcript called messenger RNA (mRNA)
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Other RNA transcripts becomes part of a complex that contains protein subunits
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For example
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Ribosomes
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Spliceosomes
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Signal recognition particles
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12-11

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Promoters are DNA sequences that “promote” gene expression
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Rate
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Start site
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Promoters are typically located just upstream of the site where transcription of a gene actually begins
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The bases in a promoter sequence are numbered in relation to the transcription start site
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Refer to Figure 12.3
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Sequence elements that play a key role in transcription
Sometimes termed the
Pribnow box, after its discoverer
Figure 12.3 The conventional numbering system of promoters
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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
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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RNA polymerase is the enzyme that catalyzes the synthesis of RNA
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In E. coli , the RNA polymerase holoenzyme is composed of
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Core enzyme
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Four subunits = a
2 bb ’
Sigma factor
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One subunit = s
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These subunits play distinct functional roles
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The RNA polymerase holoenzyme binds loosely to the DNA
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It then scans along the DNA, until it encounters a promoter region
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When it does, the sigma factor recognizes both the –35 and –10 regions
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Amino acids within the a helices hydrogen bond with bases in the promoter sequence elements
Figure 12.5
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The binding of the RNA polymerase to the promoter forms the closed complex
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Then, the open complex is formed when the
TATAAT box is unwound
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A short RNA strand is made within the open complex
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The sigma factor is released at this point
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This marks the end of initiation
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The core enzyme now slides down the DNA to synthesize an RNA strand
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Figure 12.6
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The RNA transcript is synthesized during the elongation step
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The DNA strand used as a template for RNA synthesis is termed the template or noncoding strand
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The opposite DNA strand is called the coding strand
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It has the same base sequence as the RNA transcript
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Except that T in DNA corresponds to U in RNA
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Similar to the synthesis of DNA via DNA polymerase
Figure 12.7
12-23

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Termination is the end of RNA synthesis
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It occurs when the short RNA-DNA hybrid of the open complex is forced to separate
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This releases the newly made RNA as well as the RNA polymerase
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Nuclear DNA is transcribed by three different RNA polymerases
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RNA pol I
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Transcribes all rRNA genes (except for the 5S rRNA)
RNA pol II
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Transcribes all structural genes
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Thus, synthesizes all mRNAs
Transcribes some snRNA genes
RNA pol III
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
Transcribes all tRNA genes
And the 5S rRNA gene
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Fig. 12.10a(TE Art) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Structure of a bacterial
RNA polymerase
Structure of a eukaryotic
RNA polymerase II

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Eukaryotic promoter sequences are more variable and often more complex than those of bacteria
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For structural genes, at least three features are found in most promoters
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Transcriptional start site
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TATA box
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Regulatory elements
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Refer to Figure 12.11
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Figure 12.11
Usually an adenine
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The core promoter is relatively short
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It consists of the TATA box
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Important in determining the precise start point for transcription
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The core promoter by itself produces a low level of transcription
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This is termed basal transcription
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Figure 12.11
Usually an adenine
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Regulatory elements affect the binding of RNA polymerase to the promoter
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They are of two types
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Enhancers
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Stimulate transcription
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Silencers
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Inhibit transcription
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They vary in their locations but are often found in the
–50 to –100 region
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DNA sequences that exert their effect only on nearby genes
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Example: TATA box, enhancers and silencers
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Regulatory proteins that bind to such DNA sequences
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Transcription factors
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Three categories of proteins are required for basal transcription to occur at the promoter
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RNA polymerase II
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Five different proteins called general transcription factors
(GTFs)
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A protein complex called mediator
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Figure 12.12 shows the assembly of transcription factors and RNA polymerase II at the TATA box
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Figure 12.12
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A closed complex
Figure 12.12
 TFIIH plays a major role in the formation of the open complex
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It has several subunits that perform different functions
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One subunit hydrolyzes ATP and phosphorylates a domain in RNA pol II known as the carboxyl terminal domain (CTD)
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This releases the contact between TFIIB and
RNA pol II
Other subunits act as helicases
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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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Basal transcription apparatus
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RNA pol II + the five GTFs
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The third component for transcription is a large protein complex termed mediator
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It mediates interactions between RNA pol II and various regulatory transcription factors
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Its subunit composition is complex and variable
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12-39

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The compaction of DNA to form chromatin can be an obstacle to the transcription pocess
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Most transcription occurs in interphase
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Then, chromatin is found in 30 nm fibers that are organized into radial loop domains
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Within the 30 nm fibers, the DNA is wound around histone octamers to form nucleosomes
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The histone octamer is roughly five times smaller than the complex of RNA pol II and the GTFs
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The tight wrapping of DNA within the nucleosome inhibits the function of RNA pol
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To circumvent this problem, the chromatin structure is significantly loosened during transcription
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Two common mechanisms alter chromatin structure
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1. Covalent modification of histones
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Amino terminals of histones are modified in various ways
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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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2. ATP-dependent chromatin remodeling
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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 swi tching
Mutants in SNF are s ucrose n onf ermenters
Figure 12.13
These effects may significantly alter gene expression
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Analysis of bacterial genes in the 1960s and 1970s revealed the following:
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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
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Analysis of eukaryotic structural genes in the late
1970s revealed that they are not always colinear with their functional mRNAs
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Instead, coding sequences, called exons , are interrupted by intervening sequences or introns
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Transcription produces the entire gene product
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Introns are later removed or excised
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Exons are connected together or spliced
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This phenomenon is termed RNA splicing
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It is a common genetic phenomenon in eukaryotes
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Occurs occasionally in bacteria as well
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Aside from splicing, RNA transcripts can be modified in several ways
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For example
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Trimming of rRNA and tRNA transcripts
5’ Capping and 3’ polyA tailing of mRNA transcripts
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Refer to Table 12.3
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12-47

A eukaryotic gene and its transcript

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Many nonstructural genes are initially transcribed as a large RNA
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This large RNA transcript is enzymatically cleaved into smaller functional pieces
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Figure 12.14 shows the processing of mammalian ribosomal RNA
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This processing occurs in the nucleolus
Functional RNAs that are key in ribosome structure
Figure 12.14
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Found to contain both RNA and protein subunits
However, RNA contains the catalytic ability
Therefore, it is a ribozyme
(Endonuclease)
RNase P
Endonuclease
(RNase D)
Covalently modified bases
Figure 12.15
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In the late 1970s, several research groups investigated the presence of introns in eukaryotic structural genes
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One of these groups was led by Phillip Leder
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Leder used electron microscopy to identify introns in the b
-globin gene
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It had been cloned earlier
Leder used a strategy that involved hybridization
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Double-stranded DNA of the cloned b
-globin gene is first denatured
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Then mixed with mature b
-globin mRNA
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The mRNA is complementary to the template strand of the DNA
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So the two will bind or hybridize to each other
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If the DNA is allowed to renature, this complex will prevent the reformation of the DNA double helix
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Refer to Figure 12.16
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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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The b
-globin gene from the mouse contains one or more introns
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Refer to Figure 12.17
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Figure 12.17
12-56

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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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Three different splicing mechanisms have been identified
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Group I intron splicing
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Group II intron splicing
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Spliceosome
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All three cases involve
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Removal of the intron RNA
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Linkage of the exon RNA by a phosphodiester bond
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Splicing among group I and II introns is termed self-splicing
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Splicing does not require the aid of enzymes
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Instead the RNA itself functions as its own ribozyme
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Group I and II self-splicing can occur in vitro without the additional proteins
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Figure 12.18
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In eukaryotes, the transcription of structural genes, produces a long transcript known as pre-mRNA
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Also as heterogeneous nuclear
RNA (hnRNA)
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This RNA is altered by splicing and other modifications, before it leaves the nucleus
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Splicing in this case requires the aid of a multicomponent structure known as the spliceosome
Figure 12.16
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Table 12.4 describes the occurrence of introns in genes of different species
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Most mature mRNAs have a 7-methyl guanosine covalently attached at their 5’ end
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This event is known as capping
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Cap-binding proteins play roles in the
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Movement of some RNAs into the cytoplasm
Early stages of translation
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Splicing of introns
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Figure 12.19
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Most mature mRNAs have a string of adenine nucleotides at their 3’ ends
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This is termed the polyA tail
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The polyA tail is not encoded in the gene sequence
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It is added enzymatically after the gene is completely transcribed
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The attachment of the polyA tail is shown in
Figure 12.20
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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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The spliceosome is a large complex that splices pre-mRNA
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It is composed of several subunits known as snRNPs (pronounced “snurps”)
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Each snRNP contains s mall n uclear RN A and a set of p roteins
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The subunits of a spliceosome carry out several functions
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1. Bind to an intron sequence and precisely recognize the intron-exon boundaries
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2. Hold the pre-mRNA in the correct configuration
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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 intro-exon boundaries
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The consensus sequences for the splicing of mammalian pre-mRNA are shown in Figure 12.21
Corresponds to the boxed adenine in Figure 12.22
Sequences shown in bold are highly conserved
Figure 12.21
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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Intron loops out and exons brought closer together
Figure 12.22
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Intron will be degraded and the snRNPs used again
Figure 12.22
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The biological advantage of alternative splicing is that two (or more) polypeptides can be derived from a single gene
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This allows an organism to carry fewer genes in its genome
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