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 B-galactosidase transacetylase.  The function of this enzyme is still not known, and is ignored in your book
• 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. This is also called negative control
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. This is also called positive control

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 promoter of the lac operon unless helped by a regulatory protein - catabolite activator protein (CAP)
  • Not all promoters are created equally - the promoter for the lac operon is weak - RNA polymerase will not bind very efficiently by itself
  • however, upstream from the RNA binding site is the CAP binding site
  • CAP can bind only bind to the CAP binding site when it is activated by cAMP
• CAP only becomes activated if the concentration of cyclic AMP (cAMP) is high
• Glucose inhibits the formation of cAMP by inibiting the enzyme adenylyl cyclase
  • 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 concentrations of glucose and lactose are high, the concentration of cAMP will be low, CAP will not be activated, RNA polymerase will not be able to bind well to the promoter, and the operon will be operating at a very low level (i.e. almost off)
• However, if the concentrations of glucose is low and lactose is high, the concentration of cAMP will be high, CAP will be activated and bind to the DNA which will promote RNA polymerase binding and initiate transcription

• Review of Glucose and Lactose interactions with the lac operon

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?

Gene Control in Eukaryotes

• Every cell (except gametes) have the same DNA, with the same information
• 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, can it?)
• Usually, every gene has more than one gene regulator (all of which must be on for the gene to function)

Gene Regulation in Eukaryotes

• Some of these are expressed in all cells all the time. These so-called housekeeping genes are responsible for the routine metabolic functions (e.g. respiration) common to all cells.
• Some are expressed as a cell enters a particular pathway of differentiation.
• Some are expressed all the time in only those cells that have differentiated in a particular way. For example, a plasma cell expresses continuously the gene for the antibody it synthesizes.
• Some are expressed only as conditions around and in the cell change. For example, the arrival of a hormone may turn on (or off) certain genes in that cell.

How is gene expression regulated?

There are several methods used by eukaryotes.

• The big picure

Chromatin Alteration

• Chromoomes are composed of chromatin, a complex of histone proteins and DNA
• In order to transcribe a gene, the tightly wound chromatin must become decondensed
  • One group of proteins called chromatin-remodeling complexes reshape chromatine
  • Enzymes called histone acetyl transferases (HAT) unwrap the chromatin
  • Enzymes called histone deacetylases (HDAC) condense the chromatin

Transcriptional Control 

The Promoter: RNA Polymerase binding site

Like prokaryotes, the promoter is where RNA polymerases bind (usually RNA polymerase II) to initiate transcription. RNA polymerases are enzyme complexex which reads the DNA and constructs the mRNA. Eukaryotic RNA polymerases hav a much more complex activation mechanism than prokaryotic RNA polymerase.

Steps in Initiating Transcription in Eukaryotes

• Chromatin remodeling exposes the promoter
• TATA Binding Protein (TBP) binds to the TATA box (a region of the DNA which is rich in T and A nucleotides)
• Other Basal Transcription Factors bind and interact with the promoter
• Regulatory Transcription Factors bind to enhancers, silencers, or promoter-proximal elements. This sometimes causes DNA to loop. Basal and regulatory transcription factors are collectively called the intiation complex.
• RNA Polymerase II binds to the promoter and is activated, forming a basal transcription complex.
• Transcription can begin

The Initiation Complex

• Basal Transcription Factors, such as the TATA binding protein (TBP) and TFIID (Transcription Factor II D) interact with the promoter. They are found in nearly all eukaryotic genes. They do not provide much in the way of regulation of gene transcription, but must be present for transcription to occur
• Regulatory Transcription Factors are proteins that bind to enhancers, silencers, or promoter-proximal elements. They are specific to particular genes (or families of genes) and are the chief regulatory mechanisms for gene expression in eukaryotes.

Regulatory Transcription Factors

• Regulatory Transcription Factors are proteins that bind to enhancers, silencers, or promoter-proximal elements (described below)
• These transcription factors are responsible for expression of particular genes in particular cell types and at particular stages of development. In other words, they are very particular...
• Unlike the promoter itself, the promoter-proximal elements have sequences that are unique to specific genes. In this way, they provide a mechanism for eukaryotic cells to express certain genes but not others.
• Enhancers and silencers are also gene-specific, but are often located far from the promoter

Some Eurkaryotic Regulatory Sequences are Near the Promoter

• Experiments have shown that certain proteins must bind to DNA to initiate and up-regulate gene expression
• Their role is similar to cAMP-CAP complex seen in eukaryotes
• Unlike the TATA box, the nucleotide sequence of the binding site was unique to specific genes
  • This provides a means that eukaryotes can express certain genes and not others
• Because these sequences are located close to the promoter, they are called promoter-proximal elements

Some Eukaryotic Regulatory Sequences are Far From the Promote

• Studies have shown that some regulatory sequences are tens to hundreds of thousands of bases from the promoter
• These reguatory sequences may be upstream or downstream from the promoter
• Regulatory sequences which increase the rate of transcription are called enhancers - those which decrease the rate of transcription are called silencers
• Enhancers can work if their postion is moved to a new location (but still relatively near the gene - they won't work if they are millions of base pairs away or on a different chromosome)
• Enhancers can functino if their normal 5' -- 3' orientation is flipped

Many different genes and many different types of cells share the same transcription factors - not only those that bind at the basal promoter but even some of those that bind upstream. What turns on a particular gene in a particular cell is probably the unique combination of promoter sites and the transcription factors that are chosen.  To see how this all comes together, click here.

Safe Deposit Box Analogy

Safe deposit boxes found at many banks provide a useful analogy To open any particular box in the room requires two keys: your key, whose pattern of notches fits only the lock of the box assigned to you (regulatory transcription factors), but which cannot unlock the box without a key carried by a bank employee that can activate the unlocking mechanism of any box (basal transcription factors) but cannot by itself open any box. Check out the movie "Matchstick Men" to see this in action

Hormones exert many of their effects by forming transcription factors.

The complexes of hormones with their receptor represent one class of transcription factor. Hormone "response elements", to which the complex binds, are promoter sites. Link to a discussion of these.

Just how do proteins bind to DNA?

DNA:Protein and Protein:Protein interactions are important for transcription factor function. Note modular structure of transcription factors: one part of the protein is responsible for DNA binding, another for dimer formation, another for transcriptional activation (i.e. interaction with basal transcription machinery). 

Dimer formation adds an extra element of complexity and versatility. Mixing and matching of proteins into different heterodimers and homodimers means that three distinct complexes can be formed from two proteins. 

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 Control

RNA Processing: pre-mRNA e mRNA

All the primary transcripts produced in the nucleus must undergo processing steps to produce functional RNA molecules for export to the cytosol. We shall confine ourselves to a view of the steps as they occur in the processing of pre-mRNA to mRNA.

The steps:

• Synthesis of the cap. This is a modified guanine (G) which is attached to the 5' end of the pre-mRNA as it emerges from RNA polymerase II (RNAP II). The cap protects the RNA from being degraded by enzymes that degrade RNA from the 5' end.
• Step-by-step removal of introns present in the pre-mRNA and splicing of the remaining exons. This step is required because most eukaryotic genes are split. It takes place as the pre-mRNA continues to emerge from RNAP II.
• Synthesis of the poly(A) tail. This is a stretch of adenine (A) nucleotides. When transcription is complete, the transcript is cut at a site (which may be hundreds of nucleotides before its end), and the poly(A) tail is attached to the exposed 3' end. This completes the mRNA molecule, which is now ready for export to the cytosol. (The remainder of the original transcript is degraded and the RNA polymerase leaves the DNA.)

Split Genes

• The gene for one type of collagen found in chickens is split into 52 separate exons.
• The gene for dystrophin, which is mutated in boys with muscular dystrophy, has 79 exons.
• even the genes for rRNA and tRNA are split.

The cutting and splicing of mRNA must be done with great precision. If even one nucleotide is left over from an intron or one is removed from an exon, the reading frame from that point on will be shifted, producing new codons specifying a totally different sequence of amino acids from that point to the end of the molecule (which often ends prematurely anyway when the shifted reading frame generates a STOP codon).

The removal of introns and splicing of exons is done with the spliceosome. This is a complex of several snRNA molecules and some 145 different proteins.

The introns in most pre-mRNAs begin with a GU and end with an AG. Presumably these short sequences assist in guiding the spliceosome.

Translational Control

• 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)

• mRNA molecules in cytoplasm may be degraded and recycled to make more RNA.
• This varies the amount of gene product that is produced (as a mRNA that's degraded quickly won't express much protein).

Post-Translational Control

• Protein Cleavage and/or Splicing - the newly formed protein is rarely functional as is. They typically need to be modified (i.e. insulin)

   

  

   

• Chemical modification. Protein function can be modified by addition of methyl, phosphoryl, or glycosyl groups. 
  Signal sequences direct packaging and secretion. Some proteins have "signal sequences" which direct their packaging in the Golgi and movement through the endoplasmic reticulum (ER) to be secreted. The signal sequences usually end up cleaved off.

Note: Prokaryotes exhibit transcriptional control (as seen in regulation of operons) and post-translational control (protein modification). They do not exhibit chromatin alteration (since they have naked DNA). They exhibit very little post-transcriptional control and translational control.