Molecular genetics of bacteria

1. Molecular genetics of bacteria • Emphasis: ways that bacteria differ from eukaryotes • DNA structure and function; definitions. • DNA replication • Transcription and translation • Gene regulation and regulation of metabolism • Genetic exchange among bacteria • Genetic engineering

2. DNA structure • double helix • Sugar-phosphate backbone • bases on inside, H bonded • strands anti-parallel

3. Review of directionality in DNA

4. DNA: source of cellular information • Information flow: DNARNA protein • also DNA DNA • Segments of DNA with specific information: genes. • Structure of DNA highly related to function • Information coded in sequence of bases • Complementary strands means that when each is copied, two identical molecules are made.

5. DNA in prokaryotes • Most bacteria have a single, circular, molecule of DNA • Some have more than one, some have linear DNA • Packaged w/ histone-like proteins, coiled tightly in loops, attached to membrane • 1.6 mm of DNA in a 0.002 mm sized cell. • Area of cell containing the DNA: nucleoid.

6. Plasmids • Plasmids are small, circular DNA molecules • Found in the cytoplasm of many bacteria • Plasmids are not essential for survival of the cell • They may exist singly or in many copies • Plasmids have a variety of functions • Examples: metabolic, resistance, fertility, bacteriocin, tumor-inducing, cryptic

7. DNA replication • Origin of DNA replication: particular site on DNA where copying of the DNA always starts. • Replication is bidirectional • In each direction, there is a replication fork. • Bacterial DNA is circular, so there is one Origin and one terminus • Synthesis on each DNA strand is 5’ 3’ • Replication is semi-conservative • New DNA molecules made of one old, one new strand.

8. DNA replication figures Because of requirement for 5’to 3’ synthesis, lagging strand must repeatedly top and start; needs an RNA primer each time.

9. When you’re small, you need to be stingy and quick • Look for many ways that bacteria can save energy and respond quickly to changes in environment. • E. coli needs 30 minutes to replicate its DNA, but only 20 minutes to divide into two. How? It gets a head start.

10. Methylation • Many organisms add methyl groups (-CH3) to DNA, especially to cytosine. • One of several reasons is self-protection from restriction endonucleases • Enzymes recognize sequences of nucleotides that occur at random and cut the DNA • Viral DNA injected into cytoplasm of bacteria is destroyed, protecting the bacterium. • Methylation protects bacterial DNA from cutting by its own restriction enzymes.

11. Genotype vs. Phenotype, bacterial style • Genotype: the genetic make-up of an organism. • Phenotype: the genetic information expressed by the organism. • Eukaryotes: difference is often due to masking of recessive alleles by dominant ones • In bacteria, which are generally monoploid, phenotype is determined by which genes are being expressed at the present time in response to environmental conditions.

12. About RNA 1) DNA is double stranded, but RNA is single stranded. However, RNA can base-pair with itself to create double stranded regions. RNA DNA tRNA genetics.gsk.com/graphics/ dna-big.gifhttp://www.fhi-berlin.mpg.de/th/JG/RNA.jpg http://www.santafe.edu/images/rna.gif

13. About RNA-2 2) RNA contains ribose instead of deoxyribose 3) RNA contains uracil instead of thymine. www.layevangelism.com/.../ deoxyribose.htmhttp://www.rothamsted.bbsrc.ac.uk/notebook/courses/guide/images/uracil.gif

14. 3 kinds of RNA mRNA: a copy of the gene; is translated to make protein. tRNA: smallest RNA, does actual decoding. rRNA: 3 sizes that, along with proteins, make up a ribosome. tRNA rRNA http://www.cu.lu/labext/rcms/cppe/traducti/tjpeg/trna.jpeg; Tobin and Duschek, Asking About Life; http://www.tokyo-ed.ac.jp/genet/mutation/nort.gif

15. Transcription- initiation • First step in use of the genetic info stored in DNA • RNA polymerase locates the promoter region upstream of the coding part • Sigma subunit responsible for binding RNA polymerase to promoter region of DNA, then detaches. • DNA strands separate • “Antisense strand” is copied; • Sense strand is identical to RNA except for deoxyribose and thymine instead of uracil.

16. http://cats.med.uvm.edu/cats_teachingmod/microbiology/courses/gene_regulation/images/dij.tc.elong1.jpghttp://cats.med.uvm.edu/cats_teachingmod/microbiology/courses/gene_regulation/images/dij.tc.elong1.jpg

17. The Process of Transcription-2 • RNA synthesis continues (Elongation), only one DNA strand (template) is transcribed. • RNA nucleotides, complementary to bases on DNA strand, are connected to make mRNA • Termination: must be a stop sign, right? • In bacteria, hairpin loop followed by run of U’s in the RNA. Of course, the DNA must code for complementary bases and a run of A’s. See next. Most common. OR • Termination factor “rho”. Enzyme.Forces RNA polymerase off the DNA.

18. Termination of Transcription in Bacteria The hairpin loop destabilizes the interactions between the DNA, mRNA, and polymerase; U-A basepairs are very weak, and the complex falls apart. http://www.blc.arizona.edu/marty/411/Modules/Weaver/Chap6/Fig.0649ac.gif

19. Transcription in prokaryotes • As mRNA is made, it is ready to use. • Info from more than one gene is typically found on one mRNA molecule. • Simpler process than in eukaryotes • no introns to remove • no cap or poly-A tail • no nuclear membrane to transport through • Transcription is expensive: each NTP leaves behind 2 Pi; like spending 2 ATP for every base used.

20. The Genetic Code • Four bases taken how many at a time? Need to code for 20 different amino acids. • Each base = 1 amino acid: only 4 • Every 2 bases = 1 a.a.: 16 combinations, 4 short. • Every 3 bases: 64 combinations, enough. • Every 3 bases of RNA nucleotides: codon • Each codon is complementary to 3 bases in one strand of DNA

21. Properties of the Genetic Code • Code is unambiguous: 1 codon always specifies only 1 amino acid. • Code is degenerate: although unambiguous, an amino acid can be coded for by more than one codon. • Punctuated: certain codons specify “start” and “stop”. • Universal: by viruses, both prokaryotic domains, and eukaryotes (except for some protozoa, mitochondria). • Ordered: similar codons specify the same amino acid; see especially the 1st two bases in the codon.

22. The Genetic Code-2 http://www.biology.arizona.edu/molecular_bio/problem_sets/nucleic_acids/graphics/gencode.gif

23. Bacterial ribosomes • Prokaryotic ribosomes are 70S; eukaryotic are 80S • S is Svedberg unit, how fast a particle travels during centrifugation. Affected by both mass and shape. • Large subunit: 50 S • 33 polypeptides, 5S RNA, 23 S RNA • Small subunit: 30 S • 21 polypeptides, 16S RNA • Note that 30 + 50 is not 70 • Ribosome structure and differences between prokaryotes and eukaryotes are important. • rRNAs important in taxonomy to be discussed later • Differences are the basis for success of many antibiotics

24. Translation • Literally, information translated from language of nucleotides to that of amino acids • Ribosomes (large and small subunits), mRNA, tRNAs, amino acids, and source of energy. • And various protein factors • Ribosomes attach to mRNA, read codons, tRNAs match amino acid to codon and ribosome connects amino acids to make proteins. • mRNA has start codon AUG and stop codons. • Look for animations on line

25. tRNA: the decoder a.a. attaches here anticodon http://www.designeduniverse.com/articles/Nobel_Prize/trna.jpg

26. Simultaneous transcription and translation • No processing, no nucleus; mRNA already where the ribosomes are, so they get started quickly. http://opbs.okstate.edu/~petracek/Chapter%2027%20Figures/Fig%2027-30.GIF