Chapter 6

Molecular Biology of Bacteria

OUTLINE





DNA Structure
Chromosome and Plasmids
DNA Replication
Transcription
Translation
Protein Structures and Export

Structure of Nucleotides

RNA

DNA
Fig 6.1

Hydrogen Bonding between Bases

GC paring is
stronger than AT
pairing

Fig 6.2

DNA Has Major and Minor Grooves
240°
120°
Base pairing angle
relative to backbone
creates major and minor
grooves in the 3-D DNA
structure.

Helical Structure of DNA .

7 .Thermal Denaturation of DNA Single-stranded DNA has higher absorbance at 260 nm Fig 6.

coli chromosome size: 1 mm • DNA can be overwound (positive supercoiling) or underwound (negative supercoiling). • Negative supercoiled DNA is the form predominantly found in nature.DNA Supercoiling • Supercoiling is required E. coli cell size: 1 × 2 mm E. .

Chromosomal DNA Contains Many Supercoiled Domains A nick in the DNA of one domain does not relax DNA in the other domains.8 . so damage can be localized Fig 6.

DNA Gyrase (topoisomerase II) Creates Negative Supercoiling in DNA Fig 6.9 .

Chromosome and Plasmid .

coli genome originated from horizontal transfer (distinct GC ratio.639. Many of the protein-encoding genes arose by gene duplication during evolutionary history 20% of the E.640 kbp = 4. pathogenicity islands) .221 bp 4288 protein-encoding genes (88% of the chromosome) Genetic map of E. Many genes that are highly expressed in E.64 Mbp = 4. coli (70% of the transcriptional units contain a single gene). Genes are clustered into operons. coli are oriented so that they are transcribed in the same direction that the DNA replication fork moves through them. codon distributions. coli is in minutes. but operons are NOT the rule in E.Escherichia coli Chromosome • • • • • • • • Circular 4.

10 .Escherichia coli MG1655 Chromosome Fig 6.

so plasmids should carry genes for their own replication. Inc (incompatibility) groups Plasmids can be diluted out from host cells (called curing) because they are not essential.Plasmids • • • • • Genetic elements that replicate independently of the host chromosome. Plasmids can be transferred to bacteria via conjugation (and less effectively transformation) Transfer requires a set of tra genes . Typical plasmids are circular double strand DNA molecules with the size of 3-10 kbp. Plasmid incompatibility: two closely related plasmids cannot be maintained in the same cell at the same time.

but Provide Usefulness to Host Cells • Resistance (R plasmids) Confer resistance to antibiotics e.g.) R100 • Virulence Attachment/colonization function production of virulence factors • Bacteriocins Colicins (E. coli). Nisin A (Lactic acid bacteria) • Metabolic function . Pesticins (Yersinia pestis).Plasmids Are Not Essential.

Genetic Map of R100 R100 provides resistance to several antibiotics and metal: cat: chloramphenicol str: streptomycin sul: sulfonamides tet: tetracycline mer: mercury Found in enteric bacteria Fig 6.12 .

Functions of Plasmids .

Central Dogma Replication DNA polymerase Transcription RNA polymerase Translation Ribosome .

DNA Replication .

 resulting in asymmetric leading and lagging strands. they add mononucleotides to the 3’ hydroxyl of deoxyribose and therefore elongate nucleic acid only at the 3’end. 2. . they can only extend nucleic acid chains: i.. absolutely requires a primer (made by primase).Two Important Characteristics of (all) DNA Polymerases 1.e. they cannot initiate new ones.

Five DNA Polymerases in E. coli error-prone .

Other Functions Are Required for DNA Replication (Replisome) Strand separation: •Separate and maintain single-stranded DNA (helicase and single-strand binding protein) •Handle supercoiling (DNA gyrase) Fidelity: •Ability to put correct bases. •Clamp . •Proofreading via 3’  5’ exonuclease activity Processivity: •Ability to perform multiple catalytic cycles without dissociating with the template.

16 .Events at the Replication Fork Fig 6.

18 .Joining Okazaki Fragments in the Lagging Strand Fig 6.

Bidirectional Replication DNA synthesis is bidirectional in prokaryotes Fig 6.19 Fig 6.20 .

E. However. coli grow with a doubling time of 20 min. It takes 40 min to replicate the whole E.Q. under the best condition. coli chromosome. then what is the solution? Multiple DNA replication forks .

DNA Replication and Cell Division Fig 6.21 Multiple DNA Replication Forks .

DNA Replication Is Very Accurate • Mutational rate is 10-8 – 10-11 • Two mechanisms of fidelity Correct base insertion filter by the active site Proofreading (3’  5’ exonuclease activity) .

how many point mutation(s) do you expect out of copying? A) <1 B) 1 C) ~10 D) ~100 .Q. After complete DNA replication (of the chromosome). coli from DNA replication are 108–1011 errors per base inserted. Mutation rates of E.

Per every PCR cycle. what might be the most critical property for the polymerase that is used for PCR? 4. DNA polymerase. Do we use RNA primer or DNA primer for PCR? 3.PCR (Polymerase Chain Reaction) Is Essentially DNA Replication in vitro 1. what is the maximum fold-increase in DNA copy? . What dissociate double-stranded DNA? 2.

Transcription .

Bacterial RNA Polymerase Consists of Multiple Subunits α2ββ′w + σ Crab claw shape of RNA polymerase .

the promoter is strong © 2012 Pearson Education.Sigma Factor Recognizes Where to Start Transcription A promoter is composed of two important sequences: -35 sequence -10 sequence If a promoter is close to a consensus sequence. Inc. Fig 6.26 .

Alternative Sigma Factors Recognize Different Sequences and Serve Specific Roles .

Transcription Elongation .

Rho-Independent Transcription Termination Fig 6.27 DNA-RNA interaction is significantly diminished because of the self complementary stemloop structure and the weakest A-U interactions .

Comparison of Rho-Independent and Rho-Dependent Termination .

Translation .

tRNA Is the Information Bridge Amino acid information Codon information Fig 6.33 .

tRNA Structure cloverleaf representation Fig 6.33 3D model .

34 .Aminoacyl-tRNA Synthetase Amino acid + ATP Aminoacyl-AMP + tRNA aminoacyl-AMP + PPi aminoacyl-tRNA + AMP Fig 6.

Protein Synthesis Steps Initiation mRNA binds small ribosome subunit Elongation Requires the elongation factors of EF-Tu and EF-Ts Translocation Requries the elongation factor of EF-G Termination Fig 6.35 Release factors recognize stop codons and cleave the attached polypeptide from the final tRNA .

.

Codon recognition Peptide bond formation Translocation .

what will happen? . If a ribosome reaches the end of an mRNA molecule and there is no stop codon.Q.

37 . Fig 6.Freeing Trapped (Stalled) Ribosomes tmRNA acts as both tRNA (carrying alanine) and mRNA that contains (i) codons for a peptide (susceptible to protease) and (ii) a stop codon (recruiting release factor).

23S rRNA: peptidyl transferase activity Other ribosomal RNA functions: Positioning tRNA in the A and P sites Ribosome subunit dissociation Translocation .Role of Ribosomal RNA in Protein Synthesis 16S rRNA: base pairing with the Shine-Dalgarno sequence (initiation).

Codons and Codon Bias • Codons are degenerate (redundant) 64 (4×4×4) codons for 20 amino acids.Genetic Code. . • One lysyl tRNA can bind to both AAA and AAG codons (Wobble). UAG and UGA).g. • There are three stop codons (UAA. • In an organism. but there are slight variations: e. UGA to encode tryptophan. • The genetic code is universal. • AUG (sometimes GUG or UUG) is the start codon incorporating N-formylmethionine. some codons are greatly preferred over others even though they encode the same amino acid (codon bias).

what is the third amino acid of the protein encoded by the following mRNA? 5'-CCUCAUAUGCGCCAUUAUAAGUGACACACA-3' .Genetic Code Q. Under the condition where methionine must be the first amino acid.

Incorporation of Selenocysteine and Pyrrolysine • Both amino acids are rare. • Both are encoded by stop codons (UGA and UAG. respectively) • Both have specific aminoacyl tRNA transferase • Incorporation of both rely on a recognition sequence downstream of each stop codon encoding the amino acid .

Protein Structures and Export .

Levels of Protein Structure • Primary structure Amino acid sequence • Secondary structure Depends largely on hydrogen bonding a-helix b-sheet • Tertiary structure Depends largely on hydrophobic interaction • Quaternary structure Multiple subunits .

Secondary Structure of Polypeptides .

Chaperonins Assist Protein Folding • Chaperonins = molecular chaperones • Functions Folding newly synthesized proteins (keep them from folding too abruptly…) Refolding proteins that have partially denatured .

40 .Four Key Chaperonins in E. coli Fig 6.

• Some proteins must be exported in a fully folded state (because they cannot be folded otherwise) by the Tat system. • Most proteins are exported in an unfolded state by SecA or SRP (signal recognition particle).Protein Export and Secretion Protein Export: Cytoplasm  Periplasm Protein Secretion: Cytoplasm  Outside of the cell • Signal sequence (15-20 amino acids) is required for cell membrane. . periplasmic and secreted proteins.

Export of proteins via the Major Secretory System Fig 6.41 .

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