E. coli lysed to release chromosome Fig a

General overview of bacteria One of the three major lineages of life –Eukaryotes – organisms whose cells have encased nuclei –Prokaryotes – lack a nuclear membrane Archea –1996 complete genome of Methanococcus jannaschii sequenced –More than 50% of genes completely different than bacteria and eukaryotes –Of those that are similar, genes for replication, transcription, and translation are same as eukaryotes –Genes for survival in unusual habitats similar to some bacteria Bacteria –Similar genome structure, morphology, and mechanisms of gene transfer to archea –Evolutionary biologist believe earliest single celled organism, probably prokaryote existed 3.5 billion years ago

Diversity of bacteria Outnumber all other organisms on Earth 10,000 species identified –Smallest – 200 nanometers in diameter –Largest – 500 micrometers in length (10 billion times larger than the smallest bacteria) –Habitats range from land, aquatic, to parasitic Remarkable metabolic diversity allows them to live almost anywhere

Common features of bacteria Lack defined nuclear membrane Lack membrane bound organelles Chromosomes fold to form a nucleoid body Membrane encloses cells with mesosome which serves as a source of new membranes during cell division Most have a cell wall Mucus like coating called a capsule Many move by flagella

Power of bacterial genetics is the potential to study rare events Bacteria multiply rapidly –Liquid media – E. coli grow to concentration of 109 cells per milliliter within a day –Agar media – single bacteria will multiply to 107 – 108 cells in less than a day Most studies focus on E. coli –Inhabitant of intestines in warm blooded animals –Grows without oxygen –Strains in laboratory are not pathogenic –Prototrphic – makes all the enzymes it needs for amino acid and nucleotide synthesis –Grows on minimal media containing glucose as the only carbon source –Divides about once every hour in minimal media and every 20 minutes in enriched media –Rapid multiplication make it possible to observe very rare genetic events

How to identify mutations by a genetic screen Genetic screens provide a way to observe mutations that occur very rarely such as spontaneous mutations (1 in 10 6 to 1 in 10 8 cells) –Replica plating – simultaneous transfer of thousands of colonies from one plate to another –Treatments with mutagens – increase frequency of mutations –Enrichment procedures – increase the proportion of mutant cells by killing wild-type cells –Testing for visible mutants on a petri plate

Fig. 7.12a1

Fig. 7.12a2

Fig. 7.12c1

Fig. 7.12c2

Fig. 7.12b1

Fig. 7.12b2

Fig. 8.11a

TRANSCRIPTION Transcription occurs when RNA polymerase catalyzes the 5' to 3' synthesis of an RNA molecule – a fully processive process. RNA synthesis is driven by the potential energy stored in nucleotide triphosphates and is based on matching complementary base pairs to the sequence in a template DNA strand, started at a promoter in response to signals of cell needs. You should be able to diagram (1) how transcription initiation, elongation, and termination occur in bacteria and eukaryotes and (2) how mRNAs are processed in eukaryotes.

Transcription Initiation:

Eukaryotic Genes in Pieces:

In eukaryotes, complex machines do the splicing out of the introns; in the mitochondria of tetrahymena and in phage, it is done by ribozymes that are actually part of the intron itself. There, no proteins are involved. In phage, the introns also encode so-called “homing endonucleases”.

Caps and polyA tails on eukaryotic mRNAs are involved in ribosome binding and messenger stability:

Some of the similarities and differences observed in transcription in bacteria versus eukaryotes:

How does a triplet of nucleotides specify a particular amino acid?

All of the specificity of the translational process is determined by the AA- tRNA synthase. If one puts an amino acid on and then chemically alters it to a different AA it will get inserted into the protein as if it were that initial amino acid. There are several very different families of AA- tRNA synthases that probably evolved seperately.

How Many tRNAs Are There? There are 61 different codons but only 40 or so tRNAs in most cells. The wobble hypothesis proposes that the third position of the codon can form a nonstandard base pair with the base opposite it in the tRNA anticodon. Thus, one tRNA is able to base-pair with more than one type of codon.

Many bacteria encode a suppressor tRNA that reads UGA instead of the normal codon for that particular amino acid and occasionally suppresses a nonsense (so- called amber) mutation. These were first found using T4 phage.

Is the Ribosome an Enzyme or a Ribozyme? Ribosomes contain both a number of proteins and ribosomal RNA (rRNA) and can be separated into two subunits, the large subunit and the small subunit. Three-dimensional models completed in the year 2000 revealed that the active site consists entirely of ribosomal RNA. Protein synthesis is catalyzed by RNA. The ribosome is a ribozyme—not an enzyme.

tRNAs are found on 3 sites in ribosomes: The A site is the acceptor site for the new AA-tRNA that binds to the mRNA codon. The P site holds the growing polypeptide chain. As a peptide bond forms between the amino acid on the AA-tRNA in the A site and the growing polypeptide held on the tRNA in the P site, the new A-site tRNA moves to the P site. The E site is where now-free tRNAs exit the ribosome.

Initiation Translation begins at the AUG start codon. In bacteria, this codon is preceded by a ribosome binding site (also called the Shine-Dalgarno sequence, SD) that is complementary to a short section of the 16s rRNA, in the small ribosomal subunit. In eukaryotes, this is the first AUG of a capped mRNA. In bacteria, several proteins can be encoded sequentially along the same mRNA, coordinating their production in time and space.

The SD site has some subset of the sequence 5'-AGGAGGU-3' and is located about six nucleotides upstream of the start codon.

Once the small ribosomal subunit is bound to the mRNA, the aminoacyl initiator tRNA binds to the AUG sequence. The methionine on this initiator tRNA has formic acid (COOH) attached to its amino group and is called N- formylmethionine. The large subunit binds and completes the initiation complex with the initiator tRNA is located in the P site of the ribosome. This is possible because the attached COOH blocks the positive charge of the amino group as if it were already part of a peptide chain. The formyl group, and sometimes the whole formyl methionine, is cleaved off after the protein is made.

E. coli lysed to release chromosome Fig a

General overview of bacteria One of the three major lineages of life –Eukaryotes – organisms whose cells have encased nuclei –Prokaryotes – lack a nuclear membrane Archea –1996 complete genome of Methanococcus jannaschii sequenced –More than 50% of genes completely different than bacteria and eukaryotes –Of those that are similar, genes for replication, transcription, and translation are same as eukaryotes –Genes for survival in unusual habitats similar to some bacteria Bacteria –Similar genome structure, morphology, and mechanisms of gene transfer to archea –Evolutionary biologist believe earliest single celled organism, probably prokaryote existed 3.5 billion years ago

Diversity of bacteria Outnumber all other organisms on Earth 10,000 species identified –Smallest – 200 nanometers in diameter –Largest – 500 micrometers in length (10 billion times larger than the smallest bacteria) –Habitats range from land, aquatic, to parasitic Remarkable metabolic diversity allows them to live almost anywhere

Common features of bacteria Lack defined nuclear membrane Lack membrane bound organelles Chromosomes fold to form a nucleoid body Membrane encloses cells with mesosome which serves as a source of new membranes during cell division Most have a cell wall Mucus like coating called a capsule Many move by flagella

Power of bacterial genetics is the potential to study rare events Bacteria multiply rapidly –Liquid media – E. coli grow to concentration of 109 cells per milliliter within a day –Agar media – single bacteria will multiply to 107 – 108 cells in less than a day Most studies focus on E. coli –Inhabitant of intestines in warm blooded animals –Grows without oxygen –Strains in laboratory are not pathogenic –Prototrphic – makes all the enzymes it needs for amino acid and nucleotide synthesis –Grows on minimal media containing glucose as the only carbon source –Divides about once every hour in minimal media and every 20 minutes in enriched media –Rapid multiplication make it possible to observe very rare genetic events

How to identify mutations by a genetic screen Genetic screens provide a way to observe mutations that occur very rarely such as spontaneous mutations (1 in 10 6 to 1 in 10 8 cells) –Replica plating – simultaneous transfer of thousands of colonies from one plate to another –Treatments with mutagens – increase frequency of mutations –Enrichment procedures – increase the proportion of mutant cells by killing wild-type cells –Testing for visible mutants on a petri plate