Gene Expression Is the Transformation of DNA Information Into Functional Molecules

Figure 5.22. DNA Replication. The formation of a phosphodiester bridge is catalyzed by DNA polymerases.
I. The Molecular Design of Life
5. DNA, RNA, and the Flow of Genetic Information
5.3. DNA Is Replicated by Polymerases that Take Instructions from Templates
Figure 5.23. Flow of Information from RNA to DNA in Retroviruses. The RNA genome of a retrovirus is converted
into DNA by reverse transcriptase, an enzyme brought into the cell by the infecting virus particle. Reverse transcriptase
catalyzes the synthesis of a complementary DNA strand, the digestion of the RNA, and the subsequent synthesis of the
DNA strand.
I. The Molecular Design of Life
5. DNA, RNA, and the Flow of Genetic Information
5.4. Gene Expression Is the Transformation of DNA Information Into Functional
Molecules
The information stored as DNA becomes useful when it is expressed in the production of RNA and proteins. This rich
and complex topic is the subject of several chapters later in this book, but here we introduce the basics of gene
expression. DNA can be thought of as archival information, stored and manipulated judiciously to minimize damage
(mutations). It is expressed in two steps. First, an RNA copy is made. An RNA molecule that encodes proteins can be
thought of as a photocopy of the original information it can be made in multiple copies, used, and then disposed of.
Second, an RNA molecule can be further thought of as encoding directions for protein synthesis that must be translated
to be of use. The information in messenger RNA is translated into a functional protein. Other types of RNA molecules
exist to facilitate this translation. We now examine the transcription of DNA information into RNA, the translation of
RNA information into protein, and the genetic code that links nucleotide sequence with amino acid sequence.
5.4.1. Several Kinds of RNA Play Key Roles in Gene Expression
Cells contain several kinds of RNA (Table 5.2).
1. Messenger RNA is the template for protein synthesis or translation. An mRNA molecule may be produced for each
gene or group of genes that is to be expressed in E. coli, whereas a distinct mRNA is produced for each gene in
eukaryotes. Consequently, mRNA is a heterogeneous class of molecules. In E. coli, the average length of an mRNA
molecule is about 1.2 kilobases (kb).
Kilobase (kb)
A unit of length equal to 1000 base pairs of a double-stranded
nucleic acid molecule (or 1000 bases of a single-stranded molecule).
One kilobase of double-stranded DNA has a contour length of 0.34 µ
m and a mass of about 660 kd.
2. Transfer RNA carries amino acids in an activated form to the ribosome for peptide-bond formation, in a sequence
dictated by the mRNA template. There is at least one kind of tRNA for each of the 20 amino acids. Transfer RNA
consists of about 75 nucleotides (having a mass of about 25 kd), which makes it the smallest of the RNA molecules.
3. Ribosomal RNA (rRNA) ,the major component of ribosomes, plays both a catalytic and a structural role in protein
synthesis (Section 29.3.1). In E. coli, there are three kinds of rRNA, called 23S, 16S, and 5S RNA because of their
sedimentation behavior. One molecule of each of these species of rRNA is present in each ribosome.
Ribosomal RNA is the most abundant of the three types of RNA. Transfer RNA comes next, followed by messenger
RNA, which constitutes only 5% of the total RNA. Eukaryotic cells contain additional small RNA molecules. Small
nuclear RNA (snRNA) molecules, for example, participate in the splicing of RNA exons. A small RNA molecule in the
cytosol plays a role in the targeting of newly synthesized proteins to intracellular compartments and extracellular
destinations.
5.4.2. All Cellular RNA Is Synthesized by RNA Polymerases
The synthesis of RNA from a DNA template is called transcription and is catalyzed by the enzyme RNA polymerase
(Figure 5.24). RNA polymerase requires the following components:
1. A template. The preferred template is double-stranded DNA. Single-stranded DNA also can serve as a template. RNA,
whether single or double stranded, is not an effective template; nor are RNA-DNA hybrids.
2. Activated precursors. All four ribonucleoside triphosphates
ATP, GTP, UTP, and CTP
are required.
3. A divalent metal ion. Mg2+ or Mn2+ are effective.
RNA polymerase catalyzes the initiation and elongation of RNA chains. The reaction catalyzed by this enzyme is:
The synthesis of RNA is like that of DNA in several respects (Figure 5.25). First, the direction of synthesis is 5
3.
Second, the mechanism of elongation is similar: the 3 -OH group at the terminus of the growing chain makes a
nucleophilic attack on the innermost phosphate of the incoming nucleoside triphosphate. Third, the synthesis is driven
forward by the hydrolysis of pyrophosphate. In contrast with DNA polymerase, however, RNA polymerase does not
require a primer. In addition, RNA polymerase lacks the nuclease capability used by DNA polymerase to excise
mismatched nucleotides.
All three types of cellular RNA mRNA, tRNA, and rRNA are synthesized in E. coli by the same RNA polymerase
according to instructions given by a DNA template. In mammalian cells, there is a division of labor among several
different kinds of RNA polymerases. We shall return to these RNA polymerases in Chapter 28.
5.4.3. RNA Polymerases Take Instructions from DNA Templates
RNA polymerase, like the DNA polymerases described earlier, takes instructions from a DNA template. The earliest
evidence was the finding that the base composition of newly synthesized RNA is the complement of that of the DNA
template strand, as exemplified by the RNA synthesized from a template of single-stranded φ X174 DNA (Table 5.3).
Hybridization experiments also revealed that RNA synthesized by RNA polymerase is complementary to its DNA
template. In these experiments, DNA is melted and allowed to reassociate in the presence of mRNA. RNA-DNA hybrids
will form if the RNA and DNA have complementary sequences. The strongest evidence for the fidelity of transcription
came from base-sequence studies showing that the RNA sequence is the precise complement of the DNA template
sequence (Figure 5.26).
5.4.4. Transcription Begins near Promoter Sites and Ends at Terminator Sites
RNA polymerase must detect and transcribe discrete genes from within large stretches of DNA. What marks the
beginning of a transcriptional unit? DNA templates contain regions called promoter sites that specifically bind RNA
polymerase and determine where transcription begins. In bacteria, two sequences on the 5 (upstream) side of the first
nucleotide to be transcribed function as promoter sites (Figure 5.27A). One of them, called the Pribnow box, has the
consensus sequence TATAAT and is centered at -10 (10 nucleotides on the 5 side of the first nucleotide transcribed,
which is denoted by + 1). The other, called the -35 region, has the consensus sequence TTGACA. The first nucleotide
transcribed is usually a purine.
Consensus sequence
The base sequences of promoter sites are not all identical. However,
they do possess common features, which can be represented by an
idealized consensus sequence. Each base in the consensus sequence
TATAAT is found in a majority of prokaryotic promoters. Nearly all
promoter sequences differ from this consensus sequence at only one
or two bases.
Eukaryotic genes encoding proteins have promoter sites with a TATAAA consensus sequence, called a TATA box or a
Hogness box, centered at about -25 (Figure 5.27B). Many eukaryotic promoters also have a CAAT box with a
GGNCAATCT consensus sequence centered at about -75. Transcription of eukaryotic genes is further stimulated by
enhancer sequences, which can be quite distant (as many as several kilobases) from the start site, on either its 5 or its 3
side.
RNA polymerase proceeds along the DNA template, transcribing one of its strands until it reaches a terminator sequence.
This sequence encodes a termination signal, which in E. coli is a base-paired hairpin on the newly synthesized RNA
molecule (Figure 5.28). This hairpin is formed by base pairing of self-complementary sequences that are rich in G and C.
Nascent RNA spontaneously dissociates from RNA polymerase when this hairpin is followed by a string of U residues.
Alternatively, RNA synthesis can be terminated by the action of rho, a protein. Less is known about the termination of
transcription in eukaryotes. A more detailed discussion of the initiation and termination of transcription will be given in
Chapter 28. The important point now is that discrete start and stop signals for transcription are encoded in the DNA
template.
In eukaryotes, the mRNA is modified after transcription (Figure 5.29). A "cap" structure is attached to the 5 end, and a
sequence of adenylates the poly(A) tail is added to the 3 end. These modifications will be presented in detail in Section
28.3.1.
5.4.5. Transfer RNA Is the Adaptor Molecule in Protein Synthesis
We have seen that mRNA is the template for protein synthesis. How then does it direct amino acids to become joined in
the correct sequence to form a protein? In 1958, Francis Crick wrote:
RNA presents mainly a sequence of sites where hydrogen bonding could occur. One would expect, therefore, that
whatever went onto the template in a specific way did so by forming hydrogen bonds. It is therefore a natural hypothesis
that the amino acid is carried to the template by an adaptor molecule, and that the adaptor is the part that actually fits
onto the RNA. In its simplest form, one would require twenty adaptors, one for each amino acid.
This highly innovative hypothesis soon became established as fact. The adaptor in protein synthesis is transfer RNA. The
structure and reactions of these remarkable molecules will be considered in detail in Chapter 29. For the moment, it
suffices to note that tRNA contains an amino acidattachment site and a template-recognition site. A tRNA molecule
carries a specific amino acid in an activated form to the site of protein synthesis. The carboxyl group of this amino acid
is esterified to the 3 - or 2 -hydroxyl group of the ribose unit at the 3 end of the tRNA chain (Figure 5.30). The joining of
an amino acid to a tRNA molecule to form an aminoacyl-tRNA is catalyzed by a specific enzyme called an aminoacyltRNA synthetase (or acti-vating enzyme). This esterification reaction is driven by ATP. There is at least one specific
synthetase for each of the 20 amino acids. The template-recognition site on tRNA is a sequence of three bases called an
anticodon (Figure 5.31). The anticodon on tRNA recognizes a complementary sequence of three bases, called a codon,
on mRNA.
I. The Molecular Design of Life
5. DNA, RNA, and the Flow of Genetic Information
5.4. Gene Expression Is the Transformation of DNA Information Into Functional Molecules
Table 5.2. RNA molecules in E. coli
Type
Relative amount (%) Sedimentation coefficient (S)
Ribosomal RNA (rRNA) 80
Transfer RNA (tRNA)
15
Messenger RNA (mRNA) 5
Mass (kd)
Number of nucleotides
23
1.2 × 103
3700
16
0.55 × 103
1700
5
3.6 × 101
120
4
2.5 × 101
Heterogeneous
75
I. The Molecular Design of Life
5. DNA, RNA, and the Flow of Genetic Information
5.4. Gene Expression Is the Transformation of DNA Information Into Functional Molecules
Figure 5.24. RNA Polymerase. A large enzyme comprising many subunits including β (red) and β (blue), which form
a "claw" that holds the DNA to be transcribed. The active site includes a Mg2+ ion at the center of the structure.
I. The Molecular Design of Life
5. DNA, RNA, and the Flow of Genetic Information
5.4. Gene Expression Is the Transformation of DNA Information Into Functional Molecules
Figure 5.25. Transcription Mechanism of the Chain-Elongation Reaction Catalyzed by RNA Polymerase.
I. The Molecular Design of Life
5. DNA, RNA, and the Flow of Genetic Information
5.4. Gene Expression Is the Transformation of DNA Information Into Functional Molecules
Table 5.3. Base composition (percentage) of RNA synthesized from a viral DNA template
DNA template (plus strand of φ
X174)
A
T
G
C
25
33
24
18
RNA product
25
32
23
20
U
A
C
G
I. The Molecular Design of Life
5. DNA, RNA, and the Flow of Genetic Information
5.4. Gene Expression Is the Transformation of DNA Information Into Functional Molecules
Figure 5.26. Complementarity between mRNA and DNA. The base sequence of mRNA (red) is the complement of
that of the DNA template strand (blue). The sequence shown here is from the tryptophan operon, a segment of DNA
containing the genes for five enzymes that catalyze the synthesis of tryptophan. The other strand of DNA (black) is
called the coding strand because it has the same sequence as the RNA transcript except for thymine (T) in place of uracil
(U).
I. The Molecular Design of Life
5. DNA, RNA, and the Flow of Genetic Information
5.4. Gene Expression Is the Transformation of DNA Information Into Functional Molecules
Figure 5.27. Promoter Sites for Transcription. Promoter sites are required for the initiation of transcription in both (A)
prokaryotes and (B) eukaryotes. Consensus sequences are shown. The first nucleotide to be transcribed is numbered +1.
The adjacent nucleotide on the 5 side is numbered -1. The sequences shown are those of the coding strand of DNA.
I. The Molecular Design of Life
5. DNA, RNA, and the Flow of Genetic Information
5.4. Gene Expression Is the Transformation of DNA Information Into Functional Molecules
Figure 5.28. Base Sequence of the 3 end of an mRNA transcript in E. coli. A stable hairpin structure is followed by a
sequence of uridine (U) residues.
I. The Molecular Design of Life
5. DNA, RNA, and the Flow of Genetic Information
5.4. Gene Expression Is the Transformation of DNA Information Into Functional Molecules
Figure 5.29. Modification of mRNA. Messenger RNA in eukaryotes is modified after transcription. A nucleotide "cap"
structure is added to the 5 end, and a poly(A) tail is added at the 3 end.
I. The Molecular Design of Life
5. DNA, RNA, and the Flow of Genetic Information
5.4. Gene Expression Is the Transformation of DNA Information Into Functional Molecules
Figure 5.30. Attachment of an Amino Acid to a tRNA Molecule. The amino acid (shown in blue) is esterified to the 3 hydroxyl group of the terminal adenosine of tRNA.
I. The Molecular Design of Life
5. DNA, RNA, and the Flow of Genetic Information
5.4. Gene Expression Is the Transformation of DNA Information Into Functional Molecules
Figure 5.31. Symbolic Diagram of an Aminoacyl-tRNA. The amino acid is attached at the 3 end of the RNA. The
anticodon is the template-recognition site.