1. © 2015 Pearson Education, Inc. I. The Blueprint of Life: Structure of the Bacterial Genome 4.3 Genetic Elements: Chromosomes and Plasmids

2. © 2015 Pearson Education, Inc. 4.3 Genetic Elements: Chromosomes and Plasmids Genome: entire complement of genes in cell or virus Chromosome: main genetic element in prokaryotes, usually a single piece of DNA Other genetic elements include virus genomes, plasmids, transposable elements (jumping genes) and in eukaryotes-genomes of organelles

3. © 2015 Pearson Education, Inc. 4.3 Genetic Elements: Chromosomes and Plasmids Viruses may be either RNA or DNA Plasmids: replicate separately from chromosome Great majority are double-stranded Most are circular Generally beneficial for the cell (e.g., antibiotic resistance) NOT extracellular, unlike viruses “autonomous” “episome”

4. © 2015 Pearson Education, Inc. 4.3 Genetic Elements: Chromosomes and Plasmids Chromosome is a genetic element with "housekeeping" genes Presence of essential genes is necessary for a genetic element to be called a chromosome Plasmid is a genetic element that is expendable and rarely contains genes for growth under all conditions-extra genes

5. © 2015 Pearson Education, Inc. 4.3 Genetic Elements: Chromosomes and Plasmids Transposable elements Segment of DNA that can move from one site to another site on the same or a different DNA molecule Inserted into other DNA molecules Three main types: Insertion sequences (IS) Transposons (Tn) Certain viruses (--V or –phage)

6. © 2015 Pearson Education, Inc. 4.3 Genetic Elements: Chromosomes and Plasmids The Escherichia coli chromosome Escherichia coli is a useful model organism for the study of biochemistry, genetics, and bacterial physiology The E. coli chromosome from strain MG1655 has been mapped using a combination of biological and biochemical methods (Figure 4.8)

7. © 2015 Pearson Education, Inc. Figure 4.8

8. © 2015 Pearson Education, Inc. 4.3 Genetic Elements: Chromosomes and Plasmids Some features of the E. coli chromosome Many genes encoding enzymes of a single biochemical pathway are clustered into groups called operons Operons are equally distributed on both strands ~5 Mbp in size (entire chromosome) ~40% of predicted proteins are of unknown function Average protein contains ~300 amino acids Genes are close together Few repeated genes Few intervening sequences or introns “genetic economy”

9. © 2015 Pearson Education, Inc. 4.3 Genetic Elements: Chromosomes and Plasmids Plasmids: genetic elements that replicate independently of the host chromosome (Figure 4.9) Small circular or linear DNA molecules Range in size from 1 kbp to >1 Mbp; typically less than 5% of the size of the chromosome Carry a variety of nonessential, but often very helpful, genes Abundance (copy number) is variable: relaxed vs stringent

10. © 2015 Pearson Education, Inc. Figure 4.9

11. © 2015 Pearson Education, Inc. 4.3 Genetic Elements: Chromosomes and Plasmids A cell can contain more than one plasmid Genetic information encoded on plasmids is not essential for cell function under all conditions BUT may confer a selective growth advantage under certain conditions

12. © 2015 Pearson Education, Inc. 4.3 Genetic Elements: Chromosomes and Plasmids R plasmids Resistance plasmids; confer resistance to antibiotics and other growth inhibitors (Figure 4.10) Widespread and well-studied group of plasmids Many code for conjugation functions: they can move into a new cell through conjugation

13. © 2015 Pearson Education, Inc. Figure 4.10 Plasmid R100

14. © 2015 Pearson Education, Inc. 4.3 Genetic Elements: Chromosomes and Plasmids In several pathogenic bacteria, virulence characteristics are encoded by plasmid genes Virulence factors Enable pathogen to colonize Enable pathogen to cause host damage Hemolysin Enterotoxin

15. © 2015 Pearson Education, Inc. 4.3 Genetic Elements: Chromosomes and Plasmids Bacteriocins Proteins produced by bacteria that inhibit or kill closely related species or even different strains of the same species Colicin, nisin Genes encoding bacteriocins are often carried on plasmids

16. © 2015 Pearson Education, Inc. 4.7 Transcription Transcription (DNA to RNA) is carried out by RNA polymerase using DNA as template with RNA chain growth in 5’ to 3’ direction. Transciption begins at a DNA sequence called a promoter. Also requires sigma factor or start factor. Transcription stops at specific sites called transcription terminators

17. © 2015 Pearson Education, Inc. Figure 4.20 5′5′ 3′3′ 5′5′ 3′3′ 5′5′ 3′3′ 5′5′ 3′3′ 5′5′ 5′5′ 5′5′ 3′3′ 3′3′ 5′5′ 3′3′ 5′5′ 3′3′ 5′5′ 3′3′ 5′5′ 5′5′ 5′5′ 5′5′ 3′3′ 3′3′ Transcription begins; sigma released. RNA chain grows. Sigma recognizes promoter and initiation site. RNA polymerase (core enzyme) Sigma factor Promoter region Gene(s) to be transcribed (light green strand) Sigma RNA Termination site reached; chain growth stops. Polymerase and RNA released. DNAShort transcriptsLonger transcripts

18. © 2015 Pearson Education, Inc. CCTTTTCCTTTT TTGTATTTGTAT GAGTGCGAGTGC TTTTTTTTTTTT TTTTTTTTTTTT GCCGGGGCCGGG ACCAGAACCAGA CTAGACCTAGAC AGATTAAGATTA ATATGCATATGC TGAGACTGAGAC TGTTATTGTTAT AACGCTAACGCT ATGTTTATGTTT TACACTTACACT TACTGCTACTGC ACTACGACTACG TCTAAGTCTAAG CATCTCCATCTC CTTCGACTTCGA AGGCTTAGGCTT ATCCCCATCCCC AGTTTGAGTTTG TTGCCCTTGCCC AATACCAATACC GTATACGTATAC TTTTTTTTTTTT TAACAATAACAA AGTTGAAGTTGA AAAGAAAAAGAA CGCAAACGCAAA TTTTTTTTTTTT A T C G T G A C T G C G A C G A G C G A C G A T A C T A C A C G T Sigma mRNA start –35 region Consensus Pribnow box Promoter sequence TTGAACTTTAAA 5′5′ 3′3′ 5′5′ 5′5′ 3′3′ 3′3′ 1. 2. 3. 4. 5. 6. RNA polymerase (core enzyme) Transcription Figure 4.22

19. © 2015 Pearson Education, Inc. 4.7 Transcription Termination of RNA synthesis is governed by a specific DNA sequence Intrinsic terminators: transcription is terminated without any additional factors Rho-dependent termination: Rho protein recognizes specific DNA sequences and causes a pause in the RNA polymerase

20. © 2015 Pearson Education, Inc. 4.9 Transcription in Archaea and Eukarya The Archaea contain only a single RNA polymerase Resembles eukaryotic polymerase II (Figure 4.21)

21. © 2015 Pearson Education, Inc. Figure 4.21 BacteriaArchaeaEukarya α β β'β' ω

22. © 2015 Pearson Education, Inc. 4.9 Transcription in Archaea and Eukarya Archaea have a simplified version of eukaryotic transcription apparatus Promoters and RNA polymerase similar to eukaryotes (Figure 4.26) Regulation of transcription has major similarities with Bacteria

23. © 2015 Pearson Education, Inc. 4.9 Transcription in Archaea and Eukarya Eukaryotic genes have coding and noncoding regions Exons are the coding sequences Introns are the intervening sequences Are rare in Archaea Are found in tRNA and rRNA genes of Archaea Archaeal introns excised by special endonuclease (Figure 4.27)

24. © 2015 Pearson Education, Inc. 4.9 Transcription in Archaea and Eukarya Eukaryotic RNA processing: many RNA molecules are altered before they carry out their role in the cell RNA splicing Takes place in nucleus Removes introns from RNA transcripts Performed by the spliceosome (Figure 4.28)

25. © 2015 Pearson Education, Inc. 4.9 Transcription in Archaea and Eukarya Eukaryotic RNA processing (cont'd) RNA capping (Figure 4.29) Addition of methylated guanine to 5′ end of mRNA Poly(A) tail (Figure 4.29) Addition of 100–200 adenylate residues Stabilizes mRNA and is required for translation

26. © 2015 Pearson Education, Inc. Figure 4.32 Alanine tRNA Key bases in codon: anticodon pairing mRNA 5′5′ 5′5′ 3′3′ C G G GC U Anticodon Codon Wobble position; base pairing more flexible here 3′3′ Wobble: irregular base pairing allowed at third position of tRNA (Figure 4.32) 4.11 Translation and the Genetic Code

27. © 2015 Pearson Education, Inc. 4.11 Translation and the Genetic Code 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 (e.g., mitochondria of animals, Mycoplasma, and Paramecium)

28. © 2015 Pearson Education, Inc. Figure 4.34 phe Acceptor stem D loop Acceptor end D loop Anticodon mRNA TΨC loop Codon 5′5′ 5′5′ 3′3′ 3′3′ Anticodon stem A A A A A A mA A A AA A A A A A A A C C C C C C C C mC C C C C C C CG G G G G G G G G G G G G G G G mG G U U U UU U U U U U U U Y Y Ψ T D D G UU C TΨC loop Anticodon loop Acceptor stem Acceptor end Anticodon stem Anticodon A A mGmG 3′3′ 5′5′ 4.12 Transfer RNA tRNA and cognate amino acid brought together by aminoacyl- tRNA synthetases “charged” or “loaded” tRNA

29. © 2015 Pearson Education, Inc. 4.12 Transfer RNA Fidelity of recognition process between tRNA and aminoacyl-tRNA synthetase is critical (Figure 4.35) The “Second Genetic Code”?????? Incorrect amino acid could result in a faulty or nonfunctioning protein

30. © 2015 Pearson Education, Inc. Figure 4.35 Uncharged tRNA specific for valine (tRNA Val ) Anticodon region Charged valyl tRNA, ready for protein synthesis Aminoacyl-tRNA synthetase for valine AMP Amino acid (valine) Valine Anticodon loop tRNA acceptor stem 3′3′ 5′5′ AMP CACCAC CACCAC Linkage of valine to tRNA Val Complementary shapes ensure correct reaction

31. © 2015 Pearson Education, Inc. 4.13 Protein Synthesis Polysomes: a complex formed by ribosomes simultaneously translating mRNA (Figure 4.37)

32. © 2015 Pearson Education, Inc. 4.14 Protein Folding and Secretion Once formed, a polypeptide folds to form a more stable structure. Secondary structure Interactions of the R groups force the molecule to twist and fold in a certain way (Figure 4.39) Tertiary structure Three-dimensional shape of polypeptide (Figure 4.40) Quaternary structure Number and types of polypeptides that make a protein

33. © 2015 Pearson Education, Inc. Figure 4.40 A chain α -Helix B chain β -Sheet Insulin Ribonuclease S S S S S S

34. © 2015 Pearson Education, Inc. 4.14 Protein Folding and Secretion Most polypeptides fold spontaneously into their active form Some require assistance from molecular chaperones or chaperonins for folding to occur (Figure 4.41) They only assist in the folding; they are not incorporated into protein Can also aid in refolding partially denatured proteins

35. © 2015 Pearson Education, Inc. Improperly folded protein “client” protein DnaK DnaJ Or-Transfer of improperly folded protein to GroEL/ES ATP ADP Molecular chaperone GroEL GroES Properly folded (active) protein ATP ADP Properly folded (active) protein Figure 4.41 DnaK aka Hsp70 DnaJ aka Hsp40 Hsp = heat shock protein

36. © 2015 Pearson Education, Inc. 4.14 Protein Folding and Secretion Molecular address strategy Signal sequences: found on proteins requiring transport from cell (Figure 4.42) 15–20 residues long Found at the beginning of the protein molecule Signal the cell's secretory system (Sec system) Prevent protein from completely folding

37. © 2015 Pearson Education, Inc. Figure 4.42 Translational apparatus Protein SecA Cytoplasmic membrane Periplasm Proteins with signal sequence Signal recognition particle Membrane secretion system Protein does not contain signal sequence. Ribosome mRNA Protein inserted into membrane Protein secreted into periplasm

38. © 2015 Pearson Education, Inc. 4.14 Protein Folding and Secretion Secretion of folded proteins: the Tat system Proteins that must fold in the cytoplasm are exported by a transport system distinct from Sec, called the Tat protein export system Iron–sulfur proteins Redox proteins So there are two basic secretion systems: Sec and Tat- for different types of proteins

39. © 2015 Pearson Education, Inc. 4.14 Protein Folding and Secretion Secretion of proteins: types I through VI (Figure 4.43) All are a large complex of proteins that form channels through membranes Types II and V depend on Sec or Tat Types I, III, IV, and VI do not require Sec or Tat

40. © 2015 Pearson Education, Inc. Figure 4.43 The injectisome traverses both the cytoplasmic and gram-negative outer membranes. From gram-negative bacterial cell: Cytoplasmic membrane Outer membrane Eukaryotic cytoplasmic membrane Protein, for example, a toxin Injectisome (type III secretion system) protein complex Type III secretion system-one of the more complex types Type I-VI secretion systems are additional systems that help Gram – bacteria overcome special problems