Basic Microbial Genetics part 1
Last revised: Friday, March 7, 2003
Reading: Ch. 13 in text

A. DNA (in prokaryotes)

• Every living organism has DNA = cell database. Bacteria have single chromosome (circular in all except Borrelia burgdorferi, cause of Lyme disease), no nucleus.
• View TEM of bacterium, illustrating DNA (marked n) in cell cytoplasm. Cell is a dividing Neisseria gonorrhoeae, cause of gonorrhea.
• View electron micrograph of isolated bacterial DNA
• Central dogma: information is encoded in DNA. To express this information, RNA is transcribed with same coding, then translated into amino acid sequence which folds to form active proteins.
• View animation of central dogma (Note: shockwave plug-in required)
• DNA encodes two types of molecules:
  1. database for protein structure (access by sequential transcription & translation)
  2. database for needed t-RNA, r-RNA molecules (access by transcription alone)
• Several bacterial genomes have been completely sequenced. Some chromosome sizes are listed in the table, along with sizes of yeast and human DNA for comparison.
  
  

  Organism	Domain	Chromosome size (base pairs)	Predicted polypeptide coding regions
  Mycoplasma genitalium	Bacteria	0.58 Million bp	470 proteins
  Hemophilus influenzae	Bacteria	1.83 Million bp	1740 proteins
  Methanococcus jannaschii	Archaea	1.66 Million bp	1682 proteins
  Escherichia coli	Bacteria	4.64 Million bp	4288 proteins
  Largest yeast chromosome now mapped	Eukarya	1.55 Million bp	?
  Entire yeast genome	Eukarya	15 Million bp	?
  Smallest human chromosome (Y)	Eukarya	50 Million bp	?
  Largest human chromosome (1)	Eukarya	250 Million bp	?
  Entire human genome	Eukarya	3 Billion	?

  
  
• View TIGR database of published and in progress gene sequences
• E. coli cell is often used as "model organism". Cell ~ 2 micrometers in length; has single circular DNA chromosome. Can be measured in various ways:
  1. ~1400 micrometers in length
  2. 4700 kilobase pairs
  3. 2 x 10⁹ daltons in mass
• DNA prompts many intriguing questions. For example
  1. how does cell manage a giant ball of string, which must be identically copied prior to replication, without generating hopeless tangle?
  2. Many genes are not expressed most of the time, only under certain circumstances. How does cell regulate this expression?
  3. Any damage to DNA is likely to be lethal, since there is no "backup" copy of the database. How does cell detect and repair damage?

DNA structure

• DNA made from subunits called nucleotides.
• View nucleotides (Note: Chime plug-in needed)
• Each nucleotide contains:
  1. Purine (Adenine or A, Guanine or G) or Pyrimidine (Cytosine or C, Thymine or T) bases. View purines and pyrimidines.
  2. deoxyribose sugar. View pentose sugars.
  3. 1, 2, or 3 phosphate groups. View phosphate groups.
• Nucleotides are named according to # of phosphates: e.g., dATP = deoxy adenosine triphosphate, whereas dAMP = deoxy adenosine monophosphate
• (Note: nucleotides in RNA don't have deoxyribose, don't have prefix "d"; names like ATP, ADP, AMP refer to RNA nucleotides containing ribose sugar)
• Watson & Crick discovered structure of DNA by analyzing X-ray data from Franklin & Wilkins. DNA = double helix, "backbone" consists of alternating units:
  -- deoxyribose -- phosphate -- deoxyribose -- phosphate --
  
  
  View DNA using Chime (the image directly above as a full screen image)
  
• Purine & Pyrimidine bases are attached to deoxyribose sugar, free to rotate. In DNA, form specific base pairs: A with T (2 H-bonds), G with C (3 H-bonds).
• View A-T base pairs
• View G-C base pairs
• Two chains of DNA face in opposite directions, called antiparallel (defined by which way 3' and 5' sides of sugar molecule are facing). In a linear DNA molecule, one strand has free 3'-end, other (complementary) strand has 5'-end.

  5'-CAGCTAGAGTCATCG-3'
  3'-GTCGATCTCAGTAGC-5'
  

• View DNA structure tutorial, by Eric Martz (requires Chime plug-in)
• When the two DNA strands separate, each can serve as a template for synthesis of a new complementary DNA strand. One of each of these two new, identical double stranded DNA products are separated into each of two daughter cells.

Measuring DNA amounts

• Unit of atomic weight is the dalton. A Hydrogen atom weighs 1 dalton; an Oxygen atom weights 16 daltons.
• Often refer to DNA by weight: e.g. virus DNA weighs in the range of millions of daltons, bacterial DNA weighs several billion daltons.
• Average nucleotide weight is ~ 330 daltons; one base pair has average mol. wt. of 660 daltons. Divide weight of double-stranded DNA by 660 to get approx. # of base pairs.
• Example: E. coli weighs 2.5 billion daltons. Divide by 660 = roughly 4 million base pairs
• Since most DNAs have anywhere from thousands to billions of base pairs, use units of kilobase pairs (Kbp) = 1000 bp. So E. coli in above example would have roughly 4000 Kbp (pronounced kilo base pairs).
• Another common measure is Million base pairs (Mbp). E. coli has 4 Mbp of DNA.
• Note: single-stranded RNA or DNA would be measured in Kb (kilo bases), not base pairs.

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Organization of DNA

• DNA is tightly coiled = supercoiled. Coiling is maintained by family of enzymes known as topoisomerases. Eg., DNA gyrase (topoisomerase II) induces supercoiling; topoisomerase I causes relaxation of supercoil. ATP required for supercoiling.
• View electron micrograph showing stages of supercoiling DNA
• View diagram showing supercoiled circular DNA molecule

DNA replication and repair: enzymes involved

• First enzyme isolated by Kornberg ( Nobel prize): DNA polymerase I.
  1. First Reaction:
     [dATP, dCTP, dGTP, dTTP] + DNA polymerase + Mg⁺⁺ + template DNA new DNA + P~P (pyrophosphate)
  2. Second reaction:
     P~P (+ enzyme pyrophosphatase) 2 P_i (inorganic phosphate)
• Note 1: DNA is the one molecule the cell can absolutely not afford to see broken down!!!!
  Reaction 2 is necessary to keep conc. of P~P vanishingly small; otherwise mass action law would promote slow breakdown of DNA towards equilibrium state.
• Note 2: energy for forming new sugar-phosphate bond comes from splitting a high-energy phosphate bond as P~P is removed. This always occurs at free 3'-OH group on deoxyribose (and on ribose in RNA synthesis). All nucleic acids grown by addition at 3'-end, not at 5'-end. Often referred to as 5' 3' synthesis.
• View animation of DNA replication
• View movie of DNA replication (9.6 Megabytes)
• After Kornberg's discovery, questions arose as to whether polymerase I could in fact be solely responsible for replication? Evidence suggested greater role in repair. Two more enzymes later isolated: DNA polymerase II and DNA polymerase III. Turns out DNA polymerase III is the principal replication enzyme, though polymerase I has a role also.
• Other enzymes and proteins involved:
  1. DNA helicase. This unwinds DNA just in front of opening replication fork (otherwise DNA would quickly tangle). Uses ATP, makes single-stranded cut, allows one strand to swivel freely around the other.
  2. Single-stranded DNA binding proteins. These bind to separated DNA strands, prevent from base-pairing back together
  3. RNA primase. DNA polymerase III cannot start a growing chain from scratch; needs a short primer (a few nucleotides) to add to. This is carried out by DNA-dependent RNA primase, makes very short piece of RNA by base-pairing RNA nucleotides with template DNA.
  4. DNA polymerase III. This adds new nucleotides at free 3'ends of growing chain, uses base-pairing rules to insert complementary nucleotides (A opposite T, G opposite C, etc.) Can keep on adding indefinitely for millions of nucleotides if not blockage.
  5. DNA polymerase I. This removes RNA primers, fills in gaps by base pairing, inserts new DNA nucleotides to replace RNA primer. Basically a repair enzyme, but required here.
  6. DNA ligase. A "sealing" enzyme, required to join any gaps where adjacent nucleotides on one strand have not been covalently joined. In bacteria, use phosphate bond of NAD⁺ as energy source; some ligases use ATP for energy.
• Note: many gaps result on lagging strand (see below), so lots of need for enzymes (5) and (6).

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Leading and Lagging strands

• Since two strands in DNA are antiparallel, new DNA must be synthesized in opposite directions on the two template strands.
• But overall, DNA must unwind in one direction (at replication fork), overall DNA synthesis has one direction.
• No problem for the strand growing in same direction as unwinding = leading strand. Can make one long, continuous piece of DNA
• Big problem for strand growing in opposite direction to unwinding = lagging strand; must grow away from unwinding. As new template is opened up by DNA unwinding, will have to start a new copy.
• In fact, just this situation was discovered experimentally by Okazaki; found many short DNA fragments newly synthesized from lagging strand = Okazaki fragments. Must be joined together by DNA ligase to make continuous DNA strand.
• View animation of bidirectional strand replication

Antibiotics affecting DNA synthesis

• Replication requires both uncoiling and recoiling of supercoiled DNA.
• Antibiotics of class flouroquinolones (e.g. norfloxacin) bind to DNA gyrase, inhibit restoration of supercoiling. ("protected" image.) Why is this protected?

Bidirectional synthesis of DNA

• Curious fact: E. coli can grow with generation time as low as 20 minutes at 37^o C, yet complete replication of chromosome takes much longer (even at 750-1000 base pairs per second, there's an awful lot of DNA!). How?
• Answer 1: DNA chromosome replicates bidirectionally (two replicating forks each proceeding in opposite directions). So can replicate the whole chromosome in half the time if only unidirectional synthesis. Generates "theta structures" which resemble Greek letter theta during replication. View theta structure.
  View diagram of bidirectional replication in DNA
  View Stages in replication of circular DNA.
  
• View bidirectional replication in DNA.
  But still would take ~40 minutes! How to grow cell in 20 minutes?
• Answer 2: Second round of DNA replication is initiated long before first round is finished. In exponential growth, cells can accumulate 1 or two rounds of DNA before division occurs. Possible to find up to 4 identical chromosomes in a single cell! (But not for long, cell is primed for division, eventually as nutrients diminish will wind up with only 1 chromosome per cell).
• View replicating DNA with multiple rounds of replication proceeding simultaneously.

DNA repair

• Any damage to DNA would be lethal. Cells can spend more energy repairing DNA than synthesizing it.
• Proofreading new DNA: When new DNA is synthesized, occasional errors in base pairing occur. If not corrected, could lead to mutations, loss of functions, loss of competitiveness, evolutionary weeding out. Proofreading carried out by DNA polymerases III and I; if base mismatch spotted, cut out new bases (keep track of which is template strand and which is new strand during replication), resynthesize copy strand from that neighborhood of template.
• Repairing single-strand damage. One of the most common types of damage is due to ultraviolet light (UV). Wherever two pyrimidine bases are adjacent to each other in DNA (e.g. TT), UV can cause bases to link together covalently, forming a dimer (e.g. a thymine dimer). These bases can no longer be correctly read by DNA replication enzymes (nor by transcription enzymes). If not corrected, can cause problems for cell. Same machinery as described above is used to cut out defective bases, insert new bases to repair damage. See diagram of DNA repair. ("protected" image.) Why is this protected?
• Repairing double-stranded damage. Double-stranded breaks can occur during replication if a single-stranded gap is not sealed before passing a replication fork. Also in as a result of ionizing radiation, powerful oxidizing agents, and other causes. Most bacteria can repair this damage using homologous recombination system.
• requires special set of enzymes: recA, recB, etc.
• requires a second molecule of homologous DNA. Replicating cells often contain 2 copies (preparing for division). Some cells can take up DNA from environment.
• 2 homologoues are temporarily paired in small region, and single strands are cut open and "swapped" with strand from other molecule. See (diagram part 1 and diagram part 2) ("protected" image.) Why is this protected?
• Homologous recombination can have other effects, such as introducing antibiotic resistant gene from foreign cell into a previously sensitive cell. See text.

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B. RNA (in prokaryotes)

Structure:

• Components:
  1. Ribose
  2. Phosphate
  3. Purine bases A, G; Pyrimidine bases C, U
• View comparison of RNA and DNA
• U has same base pairing properties as T (forms U=A base pairs)
• RNA is not double stranded (except in some viruses)
• RNA can have extensive "hairpin" loops
• RNA can have modified bases (after transcription) -- find unusual bases such as inosine, pseudouridine. Still have base pairing properties. Probably contribute to stability of molecule, aren't recognized by RNases which break down unmodified RNA. Messenger RNA has half-life of only 3 minutes in E. coli, so cell must constantly make new messages to make new proteins-- allows rapid adaptation to new environments.

Types of RNA

• messenger RNA -- carries codons to RNA
• ribosomal RNA -- part of ribosome structure, catalyzes peptide bond formation
• transfer RNA -- small RNAs (about 60 diff. types in E. coli), transport amino acids to ribosome for incorporation into growing polypeptides

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Transcription

• View diagram of transcription
• View animation of transcription
• View another animation of transcription
• Requires enzymeRNA polymerase
  
• opens up DNA helix for short stretch (~ 15 base pairs), selects one of two strands as template strand
• RNA synthesized in 5' to 3' direction
• Promoters: sites on DNA that are recognized as "start" signals for RNA synthesis. Typical promoter has "TATA..." base sequence (consensus sequence -- some variation) at -10 bases (upstream from actual site of RNA synthesis).
• View consensus promoters in E. coli
• role of sigma factor-- binds to RNA polymerase core enzyme, recognizes promoter. Then dissociates. View diagram of sigma factor + core enzyme.
• View model of TATA-binding protein
• Note: Bacteria can have more than one sigma factor, recognize different types of promoters.
• For example: in E. coli, when cells enter stationary phase, new sigma factor made. This allows RNA polymerase to recognize some new genes with different promoter, make RNA and then proteins which can stabilize cell during starvation, allow it to maintain quasi-dormant state until new nutrients become available.
• Another example: spore-forming cells activate different sigma factor to recognize new sets of genes involved in spore formation.
• Terminators: can be stem-loop structures with poly-U runs, or certain sequences recognized by rho factor.

Antibiotic effects

• rifamycin (prokaryotic): affects beta subunit of RNA polymerase
  View structure of rifamycin
• actinomycin: (acts on both prokaryotic and eukaryotic cells): binds to DNA at GC pairs, blocks attachment of RNA polymerase
  View structure of actinomycin

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