UNIT 7

• Gene is any DNA sequence that is transcribed into an RNA molecule
• Structural genes are encoding proteins for metabolism/ biosynthesis/ with structural role in cell
• Regulatory genes is encoding products (RNA or proteins) that interact with other DNA sequences and affect the transcription or translation of those sequences.
• Regulatory elements: DNA sequences that are not transcribed, but play a role in regulating other nucleotide sequences
• Constitutive expression: continuously expressed under normal cellular conditions – also known to be called “housekeeping genes
• DNA binding proteins have:

  • Domains: 60 to 90 amino acids responsible for binding to DNA, by forming hydrogen bonds with DNA

  • Motif: within the binding domain, a simple structure fits into the major groove of the DNA
• Gene can be defined as a single transcription unit – it contains a promoter, where the RNA polymerase enzyme binds, and the RNA coding region is then transcribed from the +1 position of transcription start site up to the transcription terminator

  ‐ Maintain above criteria but add extra regulatory elements flanking the promoter, and add the protein coding sequences for more than one structural gene between the +1 and the terminator and you will need a regulator gene
• If I draw an operon, my promoter region contains additional regulatory elements ‐ These are DNA sequences to which the regulatory protein, encoded by the regulatory gene, can bind.
• My RNA coding region is still flanked by one +1 and one terminator, however there are multiple structural genes contained within the RNA coding region
• Transcription of the operon will yield one mRNA, but it will be polycistronic – this means it contains the open reading frames or protein coding sequences for more than one protein ‐ In this example above, the mRNA will be translated into 3 different proteins.
• REGULATORY PROTEINS:

  1. Repressor proteins
  2. Activator proteins
• Repressor proteins typically bind to regulatory elements downstream of the promoter (called operators) and inhibit transcription (negative regulation).
• Activator proteins typically bind to regulatory elements upstream of the promoter (called activator binding sites) and increase transcription (positive regulation)
• Both activator and repressor proteins can exist in different conformations (allosteric proteins), the “active” conformation allows binding to DNA, the “inactive” conformation cannot bind to DNA
• Inducible operons: transcription is usually off, and needs to be turned on (i.e. it can be induced to start)
• Repressible operons: transcription is normally on, and needs to be turned off (i.e. it can be repressed to stop
• Negative control: control event (binding of regulatory protein to DNA) will inhibit gene expression
• Positive control: control event (binding of regulatory protein to DNA) will stimulate gene expression
• For a negative repressible operon, the repressor is normally inactive and not bound to the DNA, the operon is transcribed or active. Upon repression the repressor changes shape, becomes active, binds to the DNA, and prevents transcription and represses/ turns off operon expression
• For a negative inducible operon, the repressor is normally active, binds downstream of the promoter, and prevents transcription. Upon induction the repressor undergoes an allosteric shift, changes shape, is released from the DNA, and transcription is able to take place
• NEGATIVE INDUCIBLE OPERONS:

  The control at the operator site is negative. When the repressor protein is bound to the operator, transcription is inhibited.

  The transcription is inducible, so such operons are usually off (repressor bound) and need to be turned on.
• NEGATIVE INDUCIBLE OPERONS(2)

  • Inducer: small molecule that interacts with repressor, inactivates it and releases repressor from operator, allows turning on the transcription
  • these operons usually encode proteins that carry out degradative/ breakdown processes, operon induced when substrate for breakdown is present
  • The “trigger” that results in the repressor protein changing shape is typically a small molecule, called an inducer, that can interact with the repressor
• NEGATIVE REPRESSIBLE OPERONS:

  • The control at the operator site is negative. When the repressor is bound to the operator, transcription is inhibited.
  • The transcription is repressible, so such operons are usually on (repressor not bound) and needs to be turned off.
• • Corepressor: small molecule that interacts with inactive repressor, activates it and allows binding to operator, turs off transcription.
  • These operons usually encode proteins that carry out biosynthetic processes, operon repressed when there are sufficient amounts of the end product.
  • The “trigger” that results in the repressor protein changing its shape is typically a small molecule, called a co-repressor, that can interact with the inactive repressor to change its shape and make it active.
• The operon is normally on, and only repressed or turned off when there is an excess of the end product present.
• Negative repressible operon shows the inactive regulator protein not bound to the operator DNA sequence, so that RNA polymerase can access the promoter, and can travel down the template DNA to transcribe the operon and express the mRNA and protein
• Lac Operon: The lac operon encodes enzymes involved in lactose metabolism. It has three genes: lacZ encoding beta galactosidase, lacY encoding permease, and lacA encoding thiogalactoside transacetylase. LacI is the repressor protein, which binds to the operator region and prevents transcription unless lactose is absent. In the absence of lactose, the repressor binds to the operator and blocks transcription initiation. If lactose is present, it acts as an inducer, binding to the repressor and causing it to dissociate from the operator, allowing transcription to proceed.