Molecular genetics of bacteria

1Molecular 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

2DNA structure• double helix • Sugar-phosphate

backbone• bases on inside, H

bonded• strands anti-

parallel

3

Review of directionality in DNA

4DNA: source of cellular information

• Information flow: DNARNA 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.

5DNA 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.

6Plasmids

• 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

7DNA 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.

8DNA replication figures

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

9When 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.

10Methylation

• 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.

11Genotype 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.

12About RNA

genetics.gsk.com/graphics/ dna-big.gif http://www.fhi-berlin.mpg.de/th/JG/RNA.jpg

http://www.santafe.edu/images/rna.gif

1) DNA is double stranded, but RNA is single stranded.

However, RNA can base-pair with itself to create double stranded regions.

DNA

RNA

tRNA

13About RNA-2

www.layevangelism.com/.../ deoxyribose.htm http://www.rothamsted.bbsrc.ac.uk/notebook/courses/guide/images/uracil.gif

2) RNA contains ribose instead of deoxyribose

3) RNA contains uracil instead of thymine.

143 kinds of RNA

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

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.

tRNArRNA

15Transcription- 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.jpg

17The 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.

18Termination of Transcription in Bacteria

http://www.blc.arizona.edu/marty/411/Modules/Weaver/Chap6/Fig.0649ac.gif

The hairpin loop destabilizes the interactions between the DNA, mRNA, and polymerase; U-A basepairs are very weak, and the complex

falls apart.

19Transcription 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.

20The 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

21Properties 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.

22The Genetic Code-2

http://www.biology.arizona.edu/molecular_bio/problem_sets/nucleic_acids/graphics/gencode.gif

23Bacterial 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

24Translation

• 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

25tRNA: the decoder

http://www.designeduniverse.com/articles/Nobel_Prize/trna.jpg

a.a. attaches here

anticodon

26Simultaneous 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