Prescott’s Microbiology, 9th Edition
13
Bacterial Genome Replication and Expression
CHAPTER OVERVIEW
This chapter presents the basic concepts of molecular genetics: storage and organization of genetic information in the
DNA molecule. The chapter includes a description of the synthesis of RNA (transcription) and proteins (translation),
the two processes involved in gene expression. Primary emphasis is given to the genetics of bacteria.
LEARNING OUTCOMES
After reading this chapter you should be able to:
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summarize Griffith’s experiments on transformation
relate how the contributions of Avery, MacLeod, McCarty, Hershey, and Chase confirmed that DNA stores
genetic material
draw schematic representations of DNA, RNA, and amino acids that show their major features
compare and contrast the structure of DNA and RNA
identify the covalent bonds that are used to link nucleotides together to form a nucleic acid and amino acids
together to form a polypeptide
describe a bacterial replicon
summarize the events that occur during the three phases of DNA replication
create a table or concept map that illustrates the function of the major proteins found in bacterial replisome
list the enzymatic and structural elements needed by DNA polymerases for DNA synthesis
outline the major events that occur at the replication fork
draw a typical bacterial protein-coding gene, and label the important portions of the gene and the conventions
for numbering base pairs in the gene
draw a typical tRNA-and rRNA encoding gene
illustrate the organization of bacterial genes in a typical operon
describe the structure of a typical bacterial RNA polymerase holoenzyme
outline the events that occur during the three phases of transcription
discuss the role of bacterial promoters and sigma factors in transcription initiation
distinguish factor-independent termination of transcription from rho-dependent termination of transcription
explain the importance of the reading frame of a protein-coding gene
describe the universal genetic code
list deviations from the universal genetic code that have been identified in some microorganisms
explain how the wobble hypothesis enables organisms to encode fewer tRNA molecules
relate the general structure of a tRNA molecule to its role in amino activation and translation
summarize the formation of a translation initiation complex
describe the structure of bacterial ribosomes
state the initiator tRNA used by bacteria
outline the events that occur at the A, P, and E sites of the bacterial ribosome druing the elongation process of
translation
discuss the role of molecular chaperones in protein folding, and list some important examples of chaperones
describe the role of protein splicing in protein maturation
distinguish translocation of proteins from protein secretion
list bacterial translocation systems, and indicate whether they function in Gram-positive, Gram-negative, or
both types of bacteria
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Prescott’s Microbiology, 9th Edition
CHAPTER OUTLINE
I.
DNA as Genetic Material
A. Griffith (1928) demonstrated the phenomenon of transformation: live, nonvirulent bacteria could become
virulent when mixed with dead, virulent bacteria
B. Avery, MacLeod, and McCarty (1944) demonstrated that DNA was the transforming principle (the
material responsible for transformation to virulence in Griffith’s experiments)
C. Hershey and Chase (1952) showed that for the T2 bacteriophage, only DNA was needed for infectivity;
therefore, they proved that DNA was the genetic material
II. Nucleic Acid and Protein Structure
A. DNA structure
1. DNA is composed of purine and pyrimidine nucleosides that contain the sugar 2-deoxyribose and
are joined by phosphodiester bridges
2. DNA is usually a double helix consisting of two chains of nucleotides coiled around each other;
several forms of the helix exist, although the B form predominates
3. The purine adenine (A) on one strand of DNA is always paired (through hydrogen bonds) with the
pyrimidine thymine (T) on the other strand, while the purine guanine (G) is always paired with the
pyrimidine cytosine (C); thus, the two strands are said to be complementary
4. The two strands are not positioned directly opposite one another; therefore, a major groove and a
smaller minor groove are formed by the double helix backbone
5. The two polynucleotide chains are antiparallel (i.e., their sugar-phosphate backbones are oriented in
opposite directions)
B. RNA structure—RNA differs from DNA in that it is composed of the sugar ribose rather than 2deoxyribose, contains the pyrimidine uracil (U) instead of thymine, and in that it usually consists of a
single strand that can coil back on itself, rather than two strands coiled around each other
C. Protein structure—20 amino acids with different side chains (nonpolar, polar, charged) are linked by
peptide bonds to form proteins; the polypeptide has an amino or N terminal and a carboxyl or C terminal
III. DNA Replication in Bacteria
A. It is important that DNA is accurately replicated to prevent deleterious changes; although rapid (750–
1000 base pairs per second), the process has a low frequency of errors (one in 10 9 or 1010 bases)
B. DNA replication is semiconservative: each parental strand of DNA is conserved, but the two strands are
separated from each other and serve as templates for the production of new complementary daughter
strands
C. Bacterial DNA replication initiates from a single origin of replication
1. The prokaryotic chromosome is usually a replicon; that is, it typically consists of a single origin of
replication and is replicated as a unit; two replication forks (the sites of DNA synthesis) move in
opposite directions from the origin until they meet at termination sites on the opposite side; at that
point the newly synthesized chromosome is released
2. The large, linear DNA molecules of eukaryotes employ multiple replicons to efficiently replicate the
DNA within a reasonable time span
D. Replication machinery
1. DNA polymerases are enzymes that catalyze the synthesis of complementary DNA strands in the 5'
to 3' direction by adding new nucleotide monophosphates (from triphosphate substrates) to the 3'hydroxyl group of the growing chain; the enzyme needs a primer (forming a double-stranded region)
with a free 3'-hydroxyl to begin replication
2. The DNA to be replicated is bound by DnaA proteins forming the replisome, unwound by helicases,
gyrases, and topoisomerases, held unwound by single-stranded DNA binding proteins (SSBs),
primed with RNA by primase, and replicated by DNA polymerase III to create complementary
daughter strands
E. Events at the replication fork
1. In E. coli the process of replication proceeds in the following way:
a. DNA replication is initiated when DnaA protein binds to the origin of replication
2
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Prescott’s Microbiology, 9th Edition
b.
Helicases unwind the two strands of DNA and as they do so topoisomerases (e.g., DNA gyrase)
relieve the tension caused by the unwinding process; SSBs keep the single strands apart
c. Primases synthesize a small RNA molecule (approximately 10 nucleotides) that acts as a
primer for DNA synthesis
d. DNA polymerase III, like all DNA polymerases, synthesizes DNA in the 5' to 3' direction as it
creates the complementary strand of DNA according to the base-pairing rules
e. The two strands are synthesized in a different manner: on one strand (the leading strand),
synthesis is continuous, while on the other (the lagging strand), a series of fragments (Okazaki
fragments) are generated by discontinuous synthesis due to the antiparallel nature of DNA
strands and the 5' to 3' direction of polymerization
f.
DNA polymerase I removes the primers and fills the resulting gaps
g. DNA ligases join all DNA fragments to form a complete strand of DNA
2. DNA polymerase III (the product of the dnaE gene) can proofread nascent DNA chains to remove
(and replace) mismatched base pairs immediately
F. Termination of replication
1. DNA replication stops when the replisome reaches a termination site (ter); in some bacteria
replication stops when the two replication forks meet
2. The circular chromosome copies are interlocked as catenanes that are separated by topoisomerases
G. Replication of linear chromosomes
1. Linear chromosomes cannot maintain a free 3'-hydroxyl on each end (telomeres) of both DNA
strands and therefore do not provide the primer needed for DNA polymerase
2. In eukaryotes, the enzyme telomerase contains a small RNA molecule that acts as a template for the
synthesis of extensions to telomeres and allowing for DNA polymerase to work closer to the ends of
the chromosome; it is not clear how this problem is dealt with in prokaryotes that have linear
chromosomes
IV. Bacterial Gene Structure
A. Genes
1. Linear sequences of nucleotides that have a fixed start point and end point, and that encode a
polypeptide, a tRNA, or an rRNA; if it encodes a single polypeptide it is also called a cistron
2. With some exceptions, genes are not overlapping; there is a single starting point with one reading
frame in which the three-base codons are in frame with the start and stop codons
3. In prokaryotes, coding information is normally continuous although some bacterial genes are
interrupted; in eukaryotes, most genes have coding sequences (exons) that are interrupted by
noncoding sequences (introns); the mRNA transcripts in eukaryotes are spliced to remove introns
and connect exons, and alternative splicing sites may be present
B. Protein-coding genes
1. Template strand—the strand that contains coding information and directs RNA synthesis
2. Promoter—a sequence of bases that is usually situated upstream from the coding region; serves as a
recognition/binding site for RNA polymerase; different genes have different promoters, and
promoters from different species vary in sequence
3. Leader sequence—a sequence of nucleotides that is transcribed but is not translated; contains a
consensus sequence known as the Shine-Dalgarno sequence, which serves as the recognition site for
the ribosome
4. Coding region—the sequence that begins immediately downstream of the leader sequence; starts
with the template sequence 3TAC-5, which gives rise to mRNA codon 5AUG-3, the first
translated codon (specifies N-formylmethionine in bacteria, methionine in archaea and eukaryotes)
5. Trailer sequence—a transcribed but nontranslated sequence of nucleotides located immediately
downstream of the coding region and before the transcription terminator
6. Regulatory sites—sites where DNA-recognizing regulatory proteins bind to either stimulate or
inhibit gene expression (e.g., operator)
C. tRNA and rRNA genes
1. tRNA genes have promoters, leader sequences, coding regions, and trailer sequences; noncoding
regions are removed after transcription; more than one tRNA may be made from a single transcript;
the tRNAs are separated by a spacer region, which is removed after transcription
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Prescott’s Microbiology, 9th Edition
2.
rRNA genes have promoters, leader sequences, coding regions and trailer sequences; all rRNA
molecules are transcribed as a single large transcript, which is cut up after transcription, yielding the
final rRNA products
V. Transcription in Bacteria
A. Transcription is the synthesis of RNA using a DNA template
1. The RNA product is complementary to the DNA template
2. An adenine nucleotide in the DNA template directs the incorporation of a uracil nucleotide in the
RNA; otherwise, the base pair rules are the same as for DNA replication
3. Three types of RNA are produced by transcription
a. mRNA carries the message that directs the synthesis of proteins; if it carries more than one
gene transcript, it is called polycistronic or polygenic
b. tRNA molecules carry amino acids during protein synthesis
c. rRNA molecules are components of the ribosomes
B. Bacterial RNA polymerase
1. RNA polymerase (a large, multi-subunit enzyme) is responsible for the synthesis of RNA; in
bacteria, the core enzyme, consisting of four subunit types, catalyzes RNA synthesis, and the
holoenzyme includes a sigma factor that is not catalytic
2.
helps the core enzyme bind DNA at the appropriate site
C. Stages of Transcription
1. Transcription involves three separate processes: initiation, elongation, and termination
2. A promoter is the region of the DNA to which RNA polymerase binds in order to initiate
transcription; consensus sequences centered at 35 and 10 base pairs before the transcription starting
point (including the Pribnow box) are important in directing RNA polymerase to the promoter
3. During elongation, the RNA polymerase unwinds the DNA helix to create an open complex
(transcription bubble) of 16–20 base pairs within which a strand of RNA complementary to the DNA
template strand is formed in the 5' to 3' direction
4. Terminators are regions of the DNA that signal termination of the transcription process; this often
involves hairpin loops in the DNA template or the binding of rho factor protein
VI. The Genetic Code
A. For polypeptide-coding genes, the DNA base sequence corresponds to the amino acid sequence of the
polypeptide (colinearity)
B. Establishment of the genetic code—each codon that specifies a particular amino acid must be three bases
long for each of the 20 amino acids to have at least one codon; thus, the genetic code consists of 64
codons
C. Organization of the code
1. Code degeneracy—many amino acids are encoded by more than one codon
2. Start codon—the codon AUG serves as the start site for translation (protein synthesis)
3. Sense codons—61 codons that specify amino acids
4. Stop (nonsense) codons—three codons (UGA, UAG, UAA) that do not specify an amino acid, and
that are used as translation termination signals
5. Wobble—describes the somewhat loose base pairing of a tRNA anticodon to the mRNA codon;
wobble eliminates the need for a unique tRNA for each codon because the first two positions are
sufficient to establish hydrogen bonding between the mRNA and the aminoacyl-tRNAs
VII. Translation in Bacteria
A. Translation is the synthesis of a polypeptide chain directed by the nucleotide sequence in an mRNA
molecule; synthesis begins at the N-terminal and moves in the C-terminal direction
1. Ribosomes are the sites of translation
2. Polyribosomes are complexes of an mRNA molecule with several ribosomes
B. Transfer RNA and amino acid activation
1. The first stage of protein synthesis is the attachment of amino acids to tRNA molecules (catalyzed by
aminoacyl-tRNA synthetases); this process is referred to as amino acid activation
2. Each tRNA has an acceptor end (-CCA) and can only carry a specific amino acid; it also has an
anticodon triplet that is complementary to the mRNA codon triplet and leads to this specificity
C. Ribosome Structure
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Prescott’s Microbiology, 9th Edition
1. formed from 2 subunits
2. large subunit and small subunit
D. Initiation of protein synthesis
1. In bacteria, the small subunit of the ribosome binds fMet-tRNA (Met-tRNA in archaea and
eukaryotes) and then binds the mRNA at a special initiator codon (AUG); then the large subunit of
the ribosome binds
2. Three protein initiation factors also are required in prokaryotes (eukaryotes and archaea require
more initiation factors)
E. Elongation of the polypeptide chain
1. Elongation involves the sequential addition of amino acids to the growing polypeptide chain; several
polypeptide elongation factors are required for this process
2. The ribosome has three sites for binding tRNA molecules: peptidyl site (P site), aminoacyl site (A
site), and exit site (E site)
3. Each new amino acid is positioned in the A site by its tRNA, which has an anticodon that is
complementary to the codon on the mRNA molecule
4. The ribosomal enzyme peptidyl transferase catalyzes the formation of the peptide bonds between the
amino acid (or growing peptide chain) held by the tRNA in the P site and the amino acid held by the
tRNA in the A site; in doing so, the growing peptide chain is transferred to the tRNA in the A site;
the 23S rRNA is a major component of this enzyme
5. After each amino acid is added to the chain, translocation occurs, thereby moving the ribosome to
position the next codon in the A site, the tRNA carrying the growing peptide chain in the P site, and
the tRNA in the P site to the E site
F. Termination of protein synthesis
1. Takes place at any one of three nonsense codons (UAA, UAG, or UGA); three polypeptide release
factors in bacteria (one in eukaryotes and archaea) aid in the recognition of these codons; the
ribosome hydrolyzes the bond between the completed protein and the final tRNA, and the protein is
released from the ribosome, which then dissociates into its two component subunits
2. Protein synthesis is expensive, using five high-energy bonds to add one amino acid to the chain
VIII. Protein Maturation and Secretion
A. Protein folding and molecular chaperones
1. Molecular chaperones are special helper proteins that aid the nascent polypeptide in folding to its
proper shape; many have been identified and they include heat-shock proteins and stress proteins
2. Protein conformation is a direct function of amino acid sequence; proteins have self-folding,
structurally independent regions called domains; in eukaryotes, the domains fold immediately upon
synthesis, whereas in prokaryotes the domains do not fold until the complete protein is synthesized
B. Protein splicing—before folding, part of the polypeptide is removed; such splicing removes intervening
sequences (inteins) from the sequences (exteins) that remain in the final product
1. Proteins are moved outside the plasma membrane, outside the cell wall, and into the periplasmic
space to perform a variety of activities for the cell
2.
movement from the cytoplasm to the plasma membrane or periplasmic space is called translocation;
movement from the cytoplasm to outside of the cell is called secretion
C. Protein translocation and secretion in bacteria
1. Gram-positive cells secrete proteins across the plasma membrane using the Sec-dependent pathway;
gram-negative cells use this pathway and others to secrete proteins across the plasma membrane and
the outer membrane; all of these pathways require energy from ATP or GTP
2. The Sec pathway (also called the general secretion pathway) translocates proteins from the
cytoplasm across or into the plasma membrane
a. Proteins secreted by this pathway are synthesized as preproteins having a signal peptide at their
amino terminal; the signal peptide delays protein folding
b. Chaperone proteins keep the preproteins unfolded and help them reach the transport machinery
c. The protein is transferred across or into the plasma membrane; this is accompanied by the
removal of the signal peptide and subsequent folding of the protein
3. The TAT system (twin arginine translocase), in bacteria and some archaea, moves proteins across
the plasma membrane
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Prescott’s Microbiology, 9th Edition
4.
D.
Type I secretion system (ABC secretion system):
a. This pathway moves proteins from the cytoplasm across both the plasma membrane and the
outer membrane
b. Proteins secreted by this pathway contain C-terminal secretion signals
c. Proteins that comprise type I systems form channels through the membranes; translocation is
driven by both ATP hydrolysis and proton motive force
5. Type IV secretion system: can transport DNA during conjugation in addition to proteins
Protein secretion in gram-negative bacteria
1. There are six types of secretion systems in gram-negative bacteria; include the type I and type IV
secretions systems
2. Type II secretion system:
a. Observed in a number of pathogens; transports proteins that passed from the periplasmic space
across the outer membrane after the proteins pass through the plasma membrane via the Secdependent or Tat pathways
b. These systems are very complex and consist primarily of integral membrane proteins
3. Type III secretion system:
a. Used by several gram-negative pathogens to secrete virulence factors from the cytoplasm,
across both the plasma membrane and outer membrane, and into host cells
b. Some bacteria form needle-shaped type III secretion machinery and the secreted proteins are
thought to move through a translocation channel
4. Type V secretion system: starts with Sec-dependent transport across the plasma membrane, but the
proteins then form a channel in the outer membrane through which they autotransport
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Prescott’s Microbiology, 9th Edition
CRITICAL THINKING
1.
Diagram and discuss the process of semiconservative replication. Starting with a single bacterium having
only one chromosome, trace the parental DNA through three replicative cycles (resulting in eight cells).
In how many of these cells will you find DNA that was in the original parent? If the replication process
was fully conservative rather than semiconservative, how would this affect your results?
2.
Compare and contrast the transcription processes in prokaryotes and eukaryotes. Be sure to include in
your discussion the role(s) of promoters, various RNA polymerases, posttranscriptional modification,
coupled transcription/translation, and split (interrupted) genes.
3.
Comment on why utilizing a polycistronic message that encodes multiple polypeptides under a
single promoter is useful in conserving energy and allows for a balance of enzymes involved in a
particular biochemical process.
CONCEPT MAPPING CHALLENGE
Construct a concept map that describes transcription, translation, and DNA replication. Use the concepts that
follow, any other concepts or terms you need, and your own linking words between each pair of concepts in
your map.
template primer monomer peptide bond
ribosome tRNA mRNA rRNA leading strand
promoter terminator stop codon genetic code
lagging strand DNA ligase RNA polymerase
f-Met AUG codon Okazaki fragments
DNA polymerase III gyrase DNA polymerase I
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in any manner. This document may not be copied, scanned, duplicated, forwarded, distributed, or posted on a website, in whole or part.