9 week:: Molecular Biology of Bacteria (의 분자생물학)

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9th week:

Chapter 6.
Molecular Biology of Bacteria
(Bacteria의 분자생물학)
Chapter 6

Chapter 7
(12th Edition)
Molecular Biology of Bacteria
(Bacteria 의 분자생물학)
Genes and Gene Expression
(유전자와 유전자 발현)
• Functional unit of genetic information is the gene 유전자, 유전정보의 기능적
단위
• Genes in cells, composed of DNA
• Three informational macromolecules in cell
– DNA, RNA, Protein
• Genetic information flow can be divided into three stages
– Replication 복제: DNA is duplicated
– Transcription: information from DNA is transferred to RNA
• mRNA (messenger RNA): encodes polypeptides
• tRNA (transfer RNA): plays role in protein synthesis
• rRNA (ribosomal RNA): plays role in protein synthesis
– Translation: information in RNA is used to build polypeptides
• Central dogma of molecular biology
– DNA to RNA to protein
Genes and Gene Expression
(유전자와 유전자 발현)
• Eukaryotes 진핵생물: each gene is transcribed individually
• Prokaryotes 원핵생물: multiple genes may be transcribed together

 Synthesis of the
Informational Macromolecules

Source: Brock Biology of Microorganisms 12th edition.


Figure 7.1
DNA Structure (DNA 구조)
• Four nucelotides found in DNA
– Adenine (A), Guanine (G), Cytosine (C), Thymine (T)
• Backbone of DNA chain is alternating phosphates and the pentose
sugar deoxyribose
• Phosphates connect 3′- carbon of one sugar to 5 of the adjacent sugar
• All cells and some viruses have DNA in double- stranded molecule
• Two strands are antiparallel
• Two strands have complementary base sequences
– Adenine always pairs with Thymine
– Guanine always pairs with Cytosine
• Two strands form a double helix 이중나선
DNA Structure (DNA 구조)

 DNA Structure

Source: Brock Biology of Microorganisms 12th edition.


Figure 7.4
DNA Structure (DNA 구조)
• Size of DNA molecule is expressed in base pairs
• 1,000 base pairs = 1 kilobase pairs = 1 kbp
• 1,000,000 base pairs = 1 megabase pairs = 1Mbp
• E. coli genome 유전체 = 4.64 Mbp
• Each base pair takes up 0.34 nm of length along the helix
• 10 base pairs make up 1 turn of the helix

• Inverted Repeats
– Repeated sequence that is arranged in an inverse orientation
• Stem Loops
– Short double-helical regions caused by nearby inverted repeats
– Common in RNA, but not DNA
DNA Structure (DNA 구조)
• Hydrogen bonds between DNA strands hold two strands together
• Adenine–Thymine pair has two hydrogen bonds and Guanine–
Cytosine pair has three hydrogen bonds
– GC pairs are stronger than AT pairs
• High heat breaks hydrogen bonds causing denaturation (melting)
• GC-rich DNA melts at higher temperatures than AT-rich DNA

 Thermal Denaturation of DNA

Source: Brock Biology of Microorganisms 12th edition.


Figure 7.7
DNA Structure (DNA 구조)
• Supercoiled DNA: DNA is further twisted to save space
– Negative supercoiling: double helix is underwound
– Positive supercoiling: double helix is overwound
• Relaxed DNA: DNA has number of turns predicted by number of base
pairs
• Negative supercoiling is predominantly found in nature
• DNA Gyrase: introduces supercoils into DNA

• Genome 유전체: entire complement of genes in cell or virus


• Chromosome 염색체: main genetic element in prokaryotes
• Other genetic elements include virus genomes, plasmids, organelle
genomes, and transposable elements
DNA Structure (DNA 구조)
• Viruses contain either RNA or DNA genomes
– Can be linear or circular
– Can be single or double stranded
• Plasmids: replicate separately from chromosome
– Great majority are double stranded
– Most are circular
– Generally beneficial for the cell (i.e., antibiotic resistance)

• Chromosome is a genetic element with “housekeeping” genes


• Plasmid is a genetic element that is expendable and rarely contains
genes for growth under all conditions
• Presence of essential genes is necessary for a genetic element to be
called a chromosome
DNA Structure (DNA 구조)
• Transposable Elements
– Segment of DNA that can move from one site to another site on the same or
different DNA molecule
– Inserted into other DNA molecules
• Three main types
– Insertion sequences
– Transposons
– Special viruses
DNA Replication (DNA 복제)
• DNA replication is semiconservative 반보전적 복제
– Each of the two progeny double helices have one parental and one new
strand
• Precursor of each nucleotide is a deoxynucleoside 5′ triphosphate
(dNTP)
• Replication ALWAYS proceeds from the 5′ end to the 3′ end
• DNA polymerases catalyze the addition of dNTPs
• Five different DNA polymerases in E. coli
– DNA polymerase III is primary enzyme replicating chromosomal DNA
• DNA polymerases require a primer
– Primer made from RNA
DNA Replication (DNA 복제)
Overview of DNA Replication

Source: Brock Biology of Microorganisms 12th edition.


Figure 7.10
DNA Replication (DNA 복제)
• DNA synthesis begins at the origin of replication in prokaryotes
• Replication fork: zone of unwound DNA where replication occurs
• DNA helicase unwinds the DNA
• Extension of DNA
– Occurs continuously on the leading strand
– Discontinuously on the lagging strand
• Okazaki fragments are on lagging strand
DNA Replication (DNA 복제)
Events at the DNA Replication Fork

Source: Brock Biology of Microorganisms 12th edition.


Figure 7.13
DNA Replication (DNA 복제)
Sealing Two Fragments on the Lagging Strand

Source: Brock Biology of Microorganisms 12th edition.


Figure 7.15
DNA Replication (DNA 복제)
• DNA synthesis is bidirectional in prokaryotes
– Two replication forks moving in opposite directions
• DNA Pol III adds 1,000 nucleotides per second
• Replisome complex of multiple proteins involved in replication
– DNA pulled through the replisome
DNA Replication (DNA 복제)
Replication of Circular DNA: The Theta Structure

Source: Brock Biology of Microorganisms 12th edition.


Figure 7.16
DNA Replication (DNA 복제)
The Replisome

Source: Brock Biology of Microorganisms 12th edition.


Figure 7.19
DNA Replication (DNA 복제)
• DNA replication is extremely accurate
– Proofreading helps to ensure high fidelity
• Mutation rates in cells are 10-8–10-11 errors per base inserted
• Polymerase can detect mismatch through incorrect hydrogen
bonding
• Proofreading occurs in prokaryotes, eukaryotes and viral DNA
replication systems
RNA Synthesis: Transcription (전사)
• Transcription (DNA to RNA) is carried out by RNA polymerase
• RNA polymerase uses DNA as template
• RNA precursors are ATP, GTP, CTP, and UTP
• Chain growth is 5′ to 3′ just like DNA replication
• Only one of the two strands of DNA are transcribed by RNA
polymerase for any gene
• Genes are present on both strands of DNA, but at different locations
• RNA polymerase has five different subunits
• RNA polymerase recognizes DNA sites called promoters
• Promoters: site of initiation of transcription
• Promoters are recognized by sigma factor of RNA polymerase
• Transcription stops at specific sites called transcription terminators
• Unlike DNA replication, transcription involves smaller units of DNA
RNA Synthesis: Transcription (전사)
• Sigma factors recognize two highly conserved regions of promoter
• Two regions within promoters are highly conserved
– Pribnow box: located 10 bases before the start of transcription (-10 region)
– -35 region: located ~35 bases upstream of transcription
• Termination of RNA synthesis is governed by a specific DNA
sequence
– Intrinsic terminators: transcription is terminated without any additional factors
– Rho-dependant termination: Rho protein recognizes specific DNA sequences
and causes a pause in the RNA polymerase
• Unit of transcription: unit of chromosome bounded by sites where
transcription of DNA to RNA is initiated and terminated
• Most genes encode proteins, but some RNAs are not translated (i.e.,
rRNA, tRNA)
RNA Synthesis: Transcription (전사)

Transcription: Steps in RNA


Synthesis

Source: Brock Biology of Microorganisms 12th edition.


Figure 7.21
RNA Synthesis: Transcription (전사)
The Interaction of RNA Polymerase with the Promoter

Source: Brock Biology of Microorganisms 12th edition.


Figure 7.22
Protein Synthesis: Translation (번역)
• Translation: the synthesis of proteins from RNA
• Genetic code: a triplet of nucleic acid bases (codon) encodes a single
amino acid
– Specific codons for starting and stopping translation
– Degenerate code: multiple codons encode a single amino acid
– Anti-codon on tRNA recognizes codon
– Wobble: irregular base pairing allowed at third position of tRNA
• Stop codons: signal the termination of translation (UAA, UAG, and
UGA)
• Start Codon: translation begins with AUG
• Reading frame: triplet code requires translation to begin at the correct
nucleotide
• Shine-Dalgarno sequence: ensures proper reading frame
• Open Reading Frame (ORF): AUG followed by a number of codons
and a stop codon in the same reading frame
Protein Synthesis: Translation (번역)
• Codon bias: multiple codons for the same amino acid are not used
equally
– Varies with organism
– Correlated with tRNA availability
– Cloned genes from one organism may not be translated by recipient organism
because of codon bias
• Some organelles and a few cells have slight variations of the genetic
code (i.e., mitochondria of animals)
• Transfer RNA: at least one tRNA per amino acid
– Bacterial cells have 60 different tRNAs
– Mammalian cells have 100–110 different tRNAs
• Specific for both a codon and its cognate amino acid
• tRNA and amino acid brought together by aminoacyl-tRNA
synthetases
– ATP is required to attach amino acid to tRNA
• tRNA is cloverleaf in shape
Protein Synthesis: Translation (번역)
• Anti-codon: three bases of tRNA that recognize three complementary
bases on mRNA
• Fidelity of recognition process between tRNA and aminoacyl-tRNA
synthetase is critical
– Incorrect amino acid could result in a faulty/non-functioning protein
• Ribosomes: sites of protein synthesis
– Thousands of ribosomes per cell
– Composed of two subunits (30S and 50S in prokaryotes)
– S = Svedberg units
– Combination of rRNA and protein
• E. coli has 52 distinct ribosomal proteins

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