Control of Genetic Systems in Prokaryotes and Eukaryotes

Genetic Control in Prokaryotes

• Vary the numbers of specific enzymes made (regulation of gene expression)
• Slow, but can have a dramatic effect on metabolic activity
• Regulate enzymatic pathways (feedback inhibition, allosteric control)
• Rapid and can be fine-tuned, but if the enzyme system does not have this level of control, then it is useless

• Genes are grouped together based on similar functions into functional units called operons
• MANY GENES UNDER ONE CONTROL!!!
• There is one single on/off switch for the genes

lac operon in E. coli

• Function - to produce enzymes which break down lactose (milk sugar)
• lactose is not a common sugar, so there is not a great need for these enzymes
• when lactose is present, they turn on and produce enzymes
• Two components - repressor genes and functional genes
• Three functional genes:
• lacZ produces B-galactosidase.  This enzyme hydrolyzes the bond between the two sugars, glucose and galactose
• lacY produces permease.  This enzyme spans the cell membrane and brings lactose into the cell from the outside environment. The membrane is otherwise essentially impermeable to lactose.
• lacA produces acetylase.  The function of this enzyme is not known.
• Promoter (P) - aids in RNA polymerase binding
• Operator (O) - "on/off" switch - binding site for the repressor protein
• Repressor (lacI) gene
• Repressor gene (lacI) - produces repressor protein w/ two binding sites, one for the operator and one for lactose
• The repressor protein is under allosteric control - when not bound to lactose, the repressor protein can bind to the operator
• When lactose is present, an isomer of lactose, allolactose, will also be present in small amounts.  Allolactose binds to the allosteric site and changes the conformation of the repressor protein so that it is no longer capable of binding to the operator

Operation - If lactose is not present:

• the repressor gene produces repressor, which binds to the operator. This blocks the action of RNA polymerase, thereby preventing transcription.

Operation - if lactose is present:

• the repressor gene produces repressor, which has a site for binding  with allolactose.
• The allolactose/repressor compound is incapable of binding w/ the operator, so the RNA polymerase is uninhibited
• once the concentration of lactose decreases, the repressor-allolactose complex falls apart and transcription is again inhibited

The lac operon is an example of an inducible operon - it is normally off, but when a molecule called an inducer is present, the operon turns on.
The trp operon is an example of a repressible operon - it is normally on but when a molecule called a repressor is present the operon turns off.

trp Operon - and example of a repressible operon

• five genes (trpA, trpB, trpC, trpD, and trpE) involved in the production of the amino acid tryptophan
• another gene (trpR) produces an inactive repressor protein
• accumulation of the end product (tryptophan) represses synthesis of the enzymes
• tryptophan binds to the inactive repressor protein at an allosteric site
• the conformation changes and the repressor + tryptophan complex binds to the operator, repressing the operon
• tryptophan can accumulate due to internal production or from external sorces
• remember, E. coli is found in the intestines of humans so if you eat a tryptophan-rich meal, this will accumulate in the bacteria and turn off the operon
• why waste resources when a supply of this amino acid is readily available?

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It Gets More Complicated - the lac Operon Revisited

• Glucose is the sugar of choice of E. coli and if glucose is in supply, then the bacteria will preferentially break down glucose over lactose
• If glucose is present, the lac operon will be repressed - how does this happen you ask?

• RNA polymerase has a low affinity for the promter of the lac operon unless helped by a regulatory proten - cAMP receptor protein (CRP)
• CRP only becomes activated if the concentration of cyclic AMP (cAMP) is high
• Glucose inhibits the formation of cAMP
• If the concentration of glucose is high, the concentration of cAMP is low
• If the concentration of glucose is low, the concentration of cAMP is high
• Therefore, if the concentration of glucose is high, the concentration of cAMP will be low, CRP will not be activated, RNA polymerase will not be able to bind to the promoter, and the operon will be turned off
• However, if the concentration of glucose is low, the concentration of cAMP will be high, CRP will be activated and bind to the DNA which will promote RNA polymerase binding and initiate transcription
• this is assuming, of course, that lactose is present - if not, then CRP will bind but the repressor protein will be bound to the operator and the operon will still be repressed

Gene Control in Eukaryotes

• Every cell (except gametes) have the same DNA, with the same information
• This is known as genetic totipotency
• Almost all eukaryotic genes must be shut off in order to allow for cell normal function (a liver cell cannot have genes for lung cells running, not can it?)
• Usually, every gene has more than one gene regulator (all of which must be on for the gene to function)

Transcriptional

• the lac operon only has two settings: on and off
• Eukaryotic genes have numerous controls which offer a percentage increase/decease in function (i.e. successful RNA polymerase binding)

 
• Promoters - nucleotide sequences that serve as the recognition point for RNA polymerase binding
• they represent the region necessary to initiate transcription and are located immediately adjacent to the genes they regulate
• this is known as cis-regulation
• trans-regulation is regulation from afar (not adjacent to the genes)
• TATA box - an area located about 25 to 30 bases upstream (-25 to -30) from the start point of transcription
• mutaions in the TATA box severly reduce transcription
• deletions often alter the initiation point of transcription, so the TATA box is important in the proper initiation of transcription
• CAAT box - usually -70 to -80 (may be CCAAT)
• mutational anaylsis indicates that the CAAT box is crucial to the promoters ability to facilitate transcription
• mutations around the CAAT box have little to no effect on transcription
• GC box - usually about -110
• highly conserved area with GGGCGG - also crucial in transcription initiation
• Enhancers - regions of DNA which interact with regulatory proteins and can increase the efficiency of transcription initiation or activate the promoter
• The position of the enhance need not be fixed - it can be upstream, downstream or within the gene it regulates (usually within an intron - see below)
• Its orientation can be inverted without significant effect on its action
• If an enhancer is moved to another location in the genome, or if an unrelated gene is placed near an enhancer, transcription of the adjacent gene is enhanced
• Thus, they are non-specific to the given gene
• One mode of function of enhancers is to alter the configuration of the DNA by bending and looping it to bring distant enhancers and promoters into direct contact with each other in order to form complexes with transcriptions and polymerases
• Transcription Factors - proteins not associated with RNA polymerase itself but are needed for the initiation of transcription
• Diverse in nature, but several common structures are found:
• Helix-turn-helix (homeodomain) - three different planes of the helix are established and bind to the grooves of the DNA
• Zinc fingers - cystine and histidine residues bind to a Zn²⁺ ion, looping the amion acid into a finger-like chain that will rest in the grooves of DNA
• Leucine zipper - dimers result from leucine residues at every other turn of the a-helix.  When the a-helical regions form a leucine zipper, the regions beyond the zipper form a Y-shaped region that grips the DNA in a scissors-like configuration

Post-transcriptional

• The mRNA formed by transcription is called pre-mRNA
• The 5' end of the pre-mRNA is capped off with a modified form of guanine (G) forming a 5' cap before it leaves the nucleus
• helps protect the mRNA from degradation by hydrolytic enzymes
• functions as part of an "attach here" sign for ribosomes
• The 3' end of the pre-mRNA is modified by the addition of 30 to 200 adenine (A) bases forming a poly-A tail
• inhibits degredation of the mRNA
• facilitates export of mRNA from the nucleus
• When the lac operon turns on, the mRNA produces is ready to go.  Not so in eukaryotes - the pre-mRNA  is composed of numerous exons and introns.
• The introns need to be removed in order to get mature mRNA during a process called RNA splicing.
• Short nucelotide sequences are located at the ends of introns
• Particles called small nuclear ribonucleoproteins (snRNPs) recognize these splice sites
• Several different snRNP's join with additional proteins to form an even larger assembly called a spliceosome
• The spliceosome interacts with the splice sites at the end of the intron and cuts it at specific points to release then the intron and then immediately joins together the two exons that flanked the intron

Translational

• There are regions on the beginning of mRNA which do not code for proteins. These are the leaders.
• Proteins and other molecules can bind to the leader which can enhance or restrict ribosome binding (and thus translation)

Post-translational

• the newly formed protein is rarely functional as is. They typically need to be modified (i.e. insulin)