Prokaryotes

Bacterial genomes consist of nucleoids (about 4 million base pairs) and 1-50 plasmids (about 1500 base pairs) per cell. Plasmids are almost always circular and replicate independently. They commonly code for genes which confer antibiotic resistance.

4 types of genetic exchange occur among bacteria:

• transformation where bacteria uptake DNA. This does not occur frequently in nature but transformation is commonly used in the laboratory. E. Coli can take up DNA by transformation if they are made "competent" with calcium or magnesium.

• transduction is the transfer of bacterial DNA from one cell to another by means of a bacteriophage infection. Bacteriophages are viruses which infect bacteria. Their life cycle can be either lytic where they replicate and cause cell lysis or lysogenic where they integrate into the host chromosome. Transduction can either be 1) specialized where only specific genes adjacent to the site of integration are transferred along with the phage genome or 2) generalized which results from a random and accidental packaging of host DNA into the phage capsid. Thus generalized transducing particles should contain all or nearly all bacterial DNA and little or no phage DNA.

• conjugation where DNA (usually plasmids) are exchanged between bacterial cells. This is a common mechanisms of genetic exchange between bacteria. This transferred DNA can either be integrated into the recipient chromosome or stably maintained as a plasmid and passed on to daughter bacteria as an autonomously replicating unit.

• Transposons are segments of DNA which are able to move from one position to another in the genome or from the chromosomal DNA to a plasmid or the reverse. The simplest ones contain inverted repeats at each of their ends. Complex transposons usually code for antibiotic resistance and can contain transposases that excise and integrate the transposon into a new site.

Transcription

Bacteria contain a single type of RNA polymerase. In contrast to eukaryotic RNA polymerases, Bacterial RNA polymerase is able to initiate transcription in vitro without the help of additional proteins. The variable subunit of the RNA polymerase is called "sigma". There are 5 subunits which make up the RNA polymerase which is referred to as the "holoenzyme".  Each type of promoter is recognized by its own sigma subunit.

In bacteria, there are 2 sequences on the 5' (upstream) side of the first nucleotide to be transcribed which serve as promoter sites. At -10 on the DNA template there is a TATAAT sequence ("pribnow box") and there is another sequence at the -35 region. 

Unlike eukaryotes, there is no splicing or polyadenylation of the mRNA transcript in prokaryotes.

Translation

In bacteria, the mechanism for selecting a start codon is a little different since bacteria have no 5' caps. Instead, bacterial mRNA contains a specific ribosome binding site which is rich in A/G, called the Shine-Dalgarno sequence, located a few nucleotides upstream of the AUG start site of each coding region. This sequence base pairs with the 16S rRNA of the small ribosomal subunit to position the intiating AUG codon in the ribosome. This ability of the bacterial ribosome to assemble directly on a start codon AUG so long as a Shine-Dalgarno sequence precedes it means that bacterial mRNAs are often polycistronic in that they encode several different proteins, each of which is translated from the same mRNA molecule. This is not the case for eukaryotes.

Bacteria employ a rather unique trick to insure that incomplete mRNAs are not translated into proteins which could harm the cell. When the bacterial ribosome translates to the end of an incomplete RNA, a special RNA called tmRNA enters the A site of the ribosome and is itself translated into a special 11 amino acid tag to the C terminus of the truncated protein that signals to proteases that this protein should be degraded. Eucaryotes deal with this problem another way by recognizing the 5' cap and the poly A tail before translation can start.

Replication

In Procaryotes: the entire DNA replication unit is called a replicon. Procaryotic chromosomes contain 1 replicon whereas eucaryotic chromosomes contain many replicons. The protein assembly that begins DNA replication in procaryotes is referred to as a primosome.

Each replicon contains an origin of replication where DNA replication starts. In E coli, for example, this origin ("OriC") is a sequence of 245 bp which contains 3 nearly identical nucleotide sequences which are AT rich as well as 4 binding sequences further upstream for the dnaA protein which initiates the bending and unwinding of the template DNA.

Since bacteria are circular, bacteria do not have the special problems of eukaryotic DNA replication where telomeres need to be added to the chromosome end.

Gene Regulation

Bacteria avoid making enzyme(s) of a pathway when substrate is absent, but are always ready to produce these enzymes if the substrate should appear in the environment. In this way, bacterial cells are able to adapt very quickly to any change in concentration of nutrients in their environment. The primary mechanisms that bacteria have evolved to minimize the energy cost for this type of on-and-off regulation is by grouping genes that encode enyzmes of a particular pathway in a structural unit called an operon. An operon is a group of genes adjacent to one another on the bacterial chromosome which are transcribed from a single promoter as one long mRNA molecule.

Operons can be under either positive or negative control. Operons (or genes) under negative control are expressed, unless they are switched off by a repressor protein which will bind to a specific DNA sequence called the operator, making it impossible for RNA polymerase to initiate transcription at the promoter. Inversely, genes whose expression is under positive control will not be transcribed unless an active regulator protein is present which binds to a specific DNA sequence and assists the RNA polymerase in the initiation steps.

The lactose (lac) operon responsible for degradation of the sugar lactose in an inducibile operon because it functions only in the presence of an inducer (lactose here). The lac operon is also under negative regulation. In the absence of lactose, the operon is repressed by the binding of the repressor protein to the operator sequence, thus impeding the RNA polymerase function. Addition of lactose will, however, reverse this repression. (the repressor complexed with the inducer does not recognize the operator because of a conformation change in the repressor)

Full expression of the lac operon also requires a protein-mediated positive control mechanisms. In  E. coli a protein called CAP forms a complex with cAMP acquiring the ability to bind to a specific DNA sequence present in the promoter. The CAP-cAMP complex enhances binding of RNA polymerase to the promoter, thus allowing an increase in the frequency of transcription initiation.

This dual combination of positive and negative control allows E. coli to use alternative carbon sources such as lactose when glucose is absent. Falling levels of glucose induce an increase in cyclic AMP which binds to the CAP protein enabling it to bind to its specific DNA sequence near target promoters and thereby turn on the appropriate genes. But it would be wastful for CAP to induce expression of the lac operon if lactose is not present. So as a negative control, bacteria uses the lac repressor above to shut off the lac operon in the absence of lactose. The combination of both this positive and negative control acts as a type of genetic switch to make sure that the lac operon is off when lactose is not available and also that it is off when glucose is available. For the operon to be on, the cell needs -glucose and + lactose.

The tryptophan operon which contains the structural genes necessary for tryptophan biosynthesis is also under dual transcription control mechanisms. Again, it is under negative control in that an active repressor can bind to the operator blocking any transcription of the trp mRNA by the RNA polymerase. But here tryptophan is a corepressor rather than an inducer in that its presence changes the conformation of an inactive repressor protein which is then able to bind the operator. The trp operon is also under the control of an attenuation-antitermination mechanism (the leader mRNA possesses 4 repeates which can pair differently according to tryptophan availability, leading to an early termination of transcription of the operon or its full transcription.