Cycle 7 - Bacterial Gene Structures and Regulation

Question 1

Draw a bacteria gene with all the features and state their functions

Answer 1

RNA polymerase read their template 3’ to 5’

• Promoters function as double stranded DNA and sits on the template strand
  
  • -35 and -10 sequence is essential for promtoer function in bacteria
• Terminator sequence creates a hairpin loop that comp-base pairs with itself to stop transcription along with the AUAU sequence
• (not pictured) SD box is 8 bases upstream of the start codon AUG. The SD box sequence helps recruit the ribosome to the messenger RNA

Question 2

State the relationship between DNA sequence of signals and their function (ie. how would low efficiency promoters be different than high efficiency promoters?)

Answer 2

• Mutations to the -10 or -35 sequence can make promoters less or more attractive to RNA polymerase
• Terminators can also be mutated to be more or less efficient at stopping transcription (efficiency at base pairing with itself, less stable loops, longer loops) (bacteria)

Question 3

List the start and stop codons

Answer 3

• Start is TAC in the DNA template and so AUG in the RNA strand
• Stop codons are UAA, UAG, and UGA

Question 4

Explain charging of an amino acid

Answer 4

• The proper amino acid must be attached to the tRNA
• Done by enzyme aminoacyl-tRNA synthetases
  
  • They recognise the anticodon and they attach that to the proper amino acid (“charging” the tRNA)

Question 5

Describe the structure of the trp operon

Answer 5

Along the DNA of the trp operon

• trpR regulatory gene –> mRNA –> Trp repressor (inactive)
• Promoter, which RNA polymerase binds to
• The operator, site of repressor binding
• The 5 structural genes that are read by the RNA polymerase to produce mRNA –> tryptophan biosynthesis enzymes

Question 6

Describe the basic mechanism of regulation of trp operon

Answer 6

1. If trp is absent from the medium, it must be made by the cell
   
   • Nothing activates the repressor so it cannot bind to the operator
   • Thus, polymerase can freely bind to the promoter and transcription occurs
2. If trp is present in the medium, it does not need to be made by the cell
   
   • Tryptophan from the environment (a corepressor) binds to the trp repressor and activates it
   • The repressor can now bind to the operator and prevent RNA polymerase from binding to the promoter, thus transcription is not possible

Question 7

Describe repression (global, measures free amino acids) and attenuation (local, measures charged amino acids) of the trp operon

Answer 7

The trp operon is global control (transcription or no transcription)

• DNA-binding proteins (ex., trp repressor) work as dimers and create a loop in the operator that severely reduces transcription

In the region after the promoter and the operator, there is a region called the leader and the attenuator (it is in the UTR)

• Trp-charged tRNA (i.e. trp amino acid bound to tRNA) is measured by the attenuator
• Recall that translation and transcription can occur at the same time in bacteria
  
  • If the ribosome can get 2 trp codons (high trp charge tRNA), the 3/4 loop forms that prevents further transcription, even though global control OK’d the transcription
  • If the ribosome gets only 1 trp codon (low trp charge tRNA), the ribosome stalls creating the 2/3 alternate stem loop and transcription continues