All living things have a genetic molecule In prokaryotes and eukaryotes: DNA –Even in viruses, genetic material is DNA or RNA –Directs day to day operations of the cell –Provides instructions for making a new individual –passed on to daughter cells during cell division Eubacteria and Archaea differ in genome structure –Focus is on Eubacteria 1

Chromosome Structure Most bacteria genomes are single, covalently closed, circular DNA molecule –Others may have a linear molecule or several pieces DNA is negatively supercoiled –DNA is slightly underwound –Underwinding carried out by DNA gyrases –Makes separation of strands easier during transcription Supercoiling creates twisted loops –A section of supercoiled DNA is a domain –About 50 domains estimated to exist 2

3 Packaging of E. coli DNA DNA is “packaged” with proteins. Together, this is called the nucleoid. Note arrows: supercoiled DNA, when ‘nicked’, does not all unravel. Constrained by DNA packaging proteins. In Archaea, DNA packaging proteins very similar to eukaryotic histones.

Differences in DNA organization Prokaryotic DNA differs from eukaryotic Many eukaryotes have non-coding DNA = junk –Up to 90% junk in eukaryotes, not prokaryotes Eukaryotes have repeated sequences –Few repeated sequences in prokaryotes Relatively little spacer (non-coding) DNA between genes in prokaryotes Some Archaea have introns, but none in Eubacteria. Bacteria don’t waste genetic space. 4

Bacteria have transposons A bacterial genome has a dozen or so –“jumping genes”, pieces of DNA that copy themselves DNA either cuts out, inserts elsewhere or Copies itself and copy inserts elsewhere –Simple: Insertion sequences; Code for transposase and repressor –Composite transposons Insertion sequences which flank other DNA Typically antibiotic resistance genes 5

6 Insertion sequence and composite transposon From Wikipedia

7 Plasmids Plasmids: small, usually circular, independently replicating pieces of DNA with useful, not essential information. –1% to 10% of genome Types of plasmids –Fertility, resistance, catabolic, –bacteriocin, virulence, –tumor-inducing, and cryptic When is a plasmid actually part of a complex genome?

8 About plasmids-1 Fertility plasmid: genes to make a sex pilus; replicates, and a copy is passed to another cell. Resistance plasmid: genes that make the cell resistant to antibiotics, heavy metals. Catabolic plasmid: example, tol plasmid with genes for breaking down and using toluene, an organic solvent. micr302/transfer.html

9 About plasmids-2 Bacteriocin plasmid: codes for bacteriocins, proteins that kill related bacteria. Virulence plasmid: has genes needed for the bacterium to infect the host. Tumor-inducing plasmid: The Ti plasmid found in Agrobacterium tumefaciens. Codes for plant growth hormones. When the bacterium infects the plant cell, the plasmid is passed to the plant cell and the genes are expressed, causing local overgrowth of plant tissue = gall. Very useful plasmid for cloning genes into plants. Cryptic: who knows?

DNA is made up of genes A gene is a section of DNA with the information for making a protein (or an RNA) –DNA codes for rRNAs and tRNAs as well The number of genes for different types of proteins predicts ecology of the bacterium –Energy metabolism: E coli has 243 genes, Mycoplasma genitalium has only 31 M. genitalium is a parasite, depends on host Many genes are grouped in operons –Genes regulated together 10

Gene structure 11 Some sequences mark the beginning of the information, providing binding sites for proteins. This is followed by the information for making the proteins. A termination sequence signals that making mRNA should end.

12 The Genetic Code-2

13 Transcription: making mRNA RNA a polymer assembled from monomers –Ribonucleoside triphosphates: ATP, UTP, GTP,CTP RNA polymerase –Multi-component enzyme –Needs a template, but NOT a primer –In bacteria, a component (sigma) recognizes the promoter as the place on DNA to start synthesis –Synthesis proceeds 5’ to 3’, just as in DNA mRNA is complementary and antiparallel to the DNA strand being copied.

14 Transcription-2 The order of nucleotides in the RNA reflects the order in the DNA If RNA is complementary to one DNA strand, then it is identical (except for T change to U) to the other DNA strand. Either DNA strand may contain the gene! Transcription just runs the other direction.

15 Sense, antisense Compare the sense strand of the DNA to the mRNA. Note that mRNA synthesis will be 5’ to 3’ and antiparallel.

16 Sigma subunit recognizes promoter region of DNA

17 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.

Translation in bacteria: similar to eukaryotes, Except First amino acid is N-formyl methionine –Instead of methionine. Prokaryotic ribosomes differ in structure –Key for effectiveness of antibiotics Simultaneous transcription and translation –See below 18

19 Ribosome schematic

20 Translation-3 Termination –When stop codon is in A site, no tRNA binds –GTP-dependent release factor (protein) removes polypeptide from tRNA in P site. All done. –Ribosomal subunits typically dissociate. Do a Google Search for translation animation –Many hits. Note presence, absence of E site –Note shape of ribosomes –Note whether role of rRNA in catalysis is shown

21 Polysomes Multiple ribosomes attach to the mRNA and begin translating. Strings of ribosomes can be seen attached to the mRNA. Chapter%2027%20Figures/Fig% b-bottom.GIF cppe/traducti/tpoly.html

22 Simultaneous transcription and translation No processing, no nucleus; mRNA already where the ribosomes are, so they get started quickly.