Chapter 7: Blueprint of Life
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DNA: Blueprint of Life
Incredible diversity of life dictated by information within DNA
• Composed nucleotides, each containing a
nucleobase
• Adenine (A)
• Thymine (T)
• Cytosine (C)
• Guanine (G)
• 3 nucleotides encode a specific amino acid
• Amino acids make up protein
• Sequence of amino acids determines
structure and function of protein
• Structural proteins
• Enzymes
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Overview
Genome: complete set of genetic information
• Chromosome plus (technically) plasmids
• All cells have DNA genome
• Viruses may have RNA genome
• Functional unit is gene
• Encodes gene product, usually a protein
• Study of nucleotide sequence of DNA is genomics
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Overview
Cells must accomplish two tasks to multiply
• DNA replication
• Gene expression: DNA is decoded so cell can synthesize
gene products
• Transcription: information in DNA is copied into RNA
• Translation: RNA used to synthesize encoded protein
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Central Dogma of Molecular Biology
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Characteristics of DNA
DNA forms double-stranded
helix
• Carbon atoms of the
pentose sugar are
numbered
• Nucleotides joined
between 5’ PO4 and 3’ OH
• Forms sugar-phosphate
backbone
• Single DNA strand will have a
5′ end and 3′ end
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Characteristics of DNA
Strands are complementary;
held together by hydrogen
bonds between nucleobases
• Base-pairing
• Adenine (A) to thymine (T)
(two hydrogen bonds)
• Cytosine (C) to guanine (G)
(three hydrogen bonds)
• Strands are anti-parallel;
oriented in opposite
directions
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Characteristics of RNA
RNA (ribonucleic acid)
• Ribose instead of deoxyribose
• Uracil in place of thymine
• Usually shorter single strand
• Synthesized from DNA template strand
• RNA molecule is transcript
• Base-pairing rules apply except uracil pairs with adenine
• Transcript quickly separates from DNA
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Three Types of RNA
• Messenger RNA (mRNA)
• Ribosomal RNA (rRNA)
• Transfer RNA (tRNA)
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Regulating Gene Expression
Cells regulate expression of certain genes
• Rapid degradation of mRNA transcripts
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DNA Replication
DNA replication usually bidirectional from origin of replication
• Two replication forks meet at terminating site when
process complete
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Initiation of DNA Replication
DNA gyrase and helicases bind to origin of replication
• Break and unwind DNA helix
• Expose single-stranded region that can act as template
Primases synthesize short regions of RNA called primers
Requires coordinated action of many enzymes and other
components
Many of the enzymes form “assembly lines” called
replisomes
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The Process of DNA Replication
DNA polymerases synthesize in 5’ to 3’ direction
• Adds nucleotide to the 3’ end of the new strand
• DNA polymerase can only add nucleotides to existing
strand, not initiate
• Primers are required at origin of replication
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The Process of DNA Replication
Helicases “unzip” DNA strands
• Reveals template sequences
• Leading strand synthesized
continuously
• Lagging strand synthesized
discontinuously
• DNA polymerases can only add
nucleotides to 3′ end
• Production of Okazaki fragments
• Different DNA polymerase replaces
primers
• DNA ligase forms covalent bond between
adjacent nucleotides
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The Process of DNA Replication
Replication produces two complete copies of DNA
• Each daughter cell receives one
• Replication of E. coli chromosome takes approximately 40
minutes
• Optimal generation time is approximately 20 minutes
• Cell can initiate another round of replication before previous round
is complete
• Each daughter cell inherits one complete chromosome already
undergoing replication
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DNA Replication Animation
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Gene Expression in Bacteria
Transcription: RNA polymerase synthesizes single-stranded
RNA using DNA as a template
• RNA polymerase binds to sequence called a promoter
• Synthesizes in 5’ to 3’ direction
• Can initiate without primer
• Transcription stops at sequence called a terminator
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Transcription
RNA sequence is complementary, antiparallel to DNA template
strand
• DNA template is minus (−) strand
• Complement is plus (+) strand
• RNA has same sequence as (+) DNA strand except uracil
instead of thymine
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Transcription
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Transcription Animation
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Translation
Process of decoding information in mRNA to synthesize a
specific protein
• The process requires mRNA, ribosomes (rRNA), tRNAs,
and accessory proteins
• mRNA is temporary copy of genetic information
Cells must decode information in nucleotides of mRNA into
amino acids of proteins
Genetic code: three nucleotides = a codon = one amino acid
• Practically universal; with minor exceptions, it is used by
all living things
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The Genetic Code
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Translation
Initiation
Elongation
Termination
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Translation in Prokaryotes
Translation in prokaryotes begins before transcription is complete
• Once ribosome clears initiating sequences, another ribosome can
bind: forms polyribosome, or polysome
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Protein Synthesis Animation
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Differences Between Eukaryotic and Prokaryotic Gene
Expression
Eukaryotic mRNA synthesized in precursor form: pre-mRNA
• Must be processed during and after transcription
• Capping adds methylated guanine derivative to 5′ end
• Binds specific proteins: stabilize, enhance translation
• 3′ end modified via polyadenylation
• Addition of approximately 200 adenine derivatives to new 3′ end
• Poly A tail stabilizes transcript, enhances translation
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Differences Between Eukaryotic and Prokaryotic Gene
Expression
Splicing removes segments
of eukaryotic transcript
• Non-coding intervening
sequences called introns
are removed
• Expressed regions called
exons remain
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Differences Between Eukaryotic and Prokaryotic Gene
Expression
Eukaryotic mRNA produced within membrane-bound nucleus
and must be transported to cytoplasm
• Translation cannot begin during transcription
• mRNA typically monocistronic
Translation begins at first AUG
Ribosomes composed of 40s and 60s subunits
• Differences from prokaryotic ribosomes important in
targeting antibiotics
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Sensing and Responding to Environmental Fluctuations
Microorganisms constantly face changing environment and
must adapt quickly to survive
• E. coli in mammalian intestinal tract
• Alternating periods of feast and famine
• Host eats bacteria take up amino acids, vitamins, nutrients
• Bacteria shut down biosynthetic pathways, channel energy into
production of cellular components (for example, DNA, protein)
• Between meals, nutrients depleted
• Bacterial biosynthetic pathways activated
• Energy consumed, growth slowed
• Later, some E. coli eliminated in feces
• Face entirely different set of conditions
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Chorum Sensing
Allows some organisms to “sense” density of their own population
• Cells activate genes useful only when expressed by a critical
mass producing a signaling molecule
• For example , biofilm formation, pathogens′ infective process
• Some can detect or interfere with signaling molecules of other
species
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Bacterial Gene Regulation
Genes can be routinely expressed or regulated
• A set of regulated genes transcribed as single mRNA
along with its control sequences is termed operon
• For example, lac operon for lactose metabolism
• Separate operons controlled by single regulatory
mechanism constitute regulon
• Global control is simultaneous regulation of numerous genes
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Bacterial Gene Regulation
Enzymes can be grouped by type of regulation
• Constitutive enzymes synthesized constantly
• Typically indispensable roles in central metabolism (for example, enzymes
of glycolysis)
• Inducible enzymes synthesized only when needed (for
example, β-galactosidase turned on only when lactose present)
• Avoids waste of resources
• Repressible enzymes
produced routinely, but are
turned off when not needed
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Mechanisms to Control Gene Regulation
Mechanisms to Control Transcription
• Must be readily reversible, allow cells to control relative
number of transcripts produced
• Two most common regulatory mechanisms are alternative
sigma factors and DNA-binding proteins
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Alternative Sigma Factors
Standard sigma factor is loose component of RNA
polymerase; recognizes promoters for genes expressed
during routine growth conditions
Alternative sigma factors recognize different sets of
promoters to control expression of specific groups of genes
• For example, sporulation in Bacillus subtilis controlled by
multiple different alternative sigma factors
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DNA Binding Proteins
Can act as repressors
• Repressor blocks transcription (negative regulation)
• Binds to operator, stops RNA polymerase
• Repressors are allosteric: have binding site that alters ability to bind
to DNA
Repressors can play a role in two types of transcriptional
regulatory systems:
• Induction: repressor binds to operator, blocks transcription
• Inducer binds to repressor, repressor unable to bind
• Repression: repressor cannot bind to operator
• Corepressor attaches to repressor, complex now binds to operator
and blocks transcription
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Transcriptional Regulation by Repressors
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DNA Binding Proteins
Can act as activators
• Activator facilitates transcription (positive regulation)
• Ineffective promoter preceded by activator-binding site
• Binding of activator enhances ability of RNA polymerase to initiate
transcription at promoter
• Inducer binding to activator allows binding to DNA
• Inducer may also interfere with repressor
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Regulatory Proteins Animation
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The lac Operon as a Model
Encodes proteins involved with transport and degradation of
lactose
• Turned on when glucose is not available, but lactose is
• Turned off when glucose is available
• Cell uses most efficiently metabolized carbon source
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Lactose and the lac Operon
When lactose is not available
• Repressor prevents transcription;
binds operator
When lactose is present
• Some converted to inducer
allolactose; binds repressor
• Repressor releases
operator
• RNA polymerase transcribes
operon
• Only occurs when glucose is not
available
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Glucose and the lac Operon
When both glucose and lactose are present
• Carbon catabolite repression (CCR) prevents expression
of genes that metabolize lactose in presence of glucose
• Prioritize carbon/energy sources; yields diauxic growth
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Lac operon Animation
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Eukaryotic Gene Regulation
Eukaryotic regulation more complicated than in prokaryotes
Variety of control methods:
• Modification of chromosome structure
• Regulation of initiation of transcription
• Altering pre-mRNA processing and modification
• RNA interference (RNAi)
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RNA Interference (RNAi)
Short RNA strand joins multi-protein unit
• RNA-induced silencing complex
(RISC)
RNA strand serves as probe for binding
to mRNA
Tags mRNA for destruction
• Rapidly silences transcripts
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