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2.7: Transcriptional Regulation: Transcription termination, the trp operon

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  • Transcription termination in prokaryotes

    • Because protein coding regions are closely spaced in the genomes of microorganisms, independent control of neighboring genes is possible only if a transcription termination site lies between them (i.e. downstream genes must be independently regulated).
    • There are two general mechanisms of transcription termination:
    1. one requires the presence of a transcription termination protein called rho,
    2. the other requires no associated proteins.

    Rho-independent termination

    • Rho-independent transcription termination sequences have two general characteristic features:
    1. A 3' stretch of T residues
    2. A 'GC' rich interrupted palindrome just upstream of the 3' poly T region.

    Screenshot (268).png

    Figure 2.7.1: Rho-independent transcription termination site

    The important features of Rho-independent transcription termination are as follows:

    1. The inverted repeat region can self-hybridize to form a "stem-loop" structure:

    Screenshot (269).png

    Figure 2.7.2: Stem-loop structure

    2. The GC rich stem loop structure interacts with RNA polymerase to cause it to pause.

    3. The short UUU region, which is base pairing with AAA sequence on the anti-sense DNA strand, has low thermal stability and melts - releasing the nascent RNA transcript.

    Attenuation at the trp operon and premature mRNA chain termination

    • The trp operon is regulated by the trp repressor protein.
      • Trp repressor protein binds to tryptophan and undergoes a conformational change which allows it to bind to the trp operator region (downstream of the promoter).
      • This prevents transcription of the trp operon in the presence of tryptophan (i.e. tryptophan represses expression).
      • However the repression is somewhat incomplete and there is a second mechanism which contributes to transcription repression.
    • When tryptophan is present, and the operon is repressed, the few transcripts which are made are actually quite short and do not encompass the entire polycistronic message of the trp operon.
    • The short mRNA that is made comprises only a region called the leader sequence:

    Screenshot (270).png

    Figure 2.7.3: Leader sequence

    • The leader region contains an attenuator sequence - a site where a choice is made between elongation of the growing trp transcript or (premature) termination.
    • Attenuation depends on the interplay between ribosome binding and translation of the nascent mRNA transcript and the formation of a particular stem-loop structure in the mRNA leader sequence.
      • Formation of this structure depends on the rate of ribosomal translation of the leader sequence (the leader sequence contains an AUG start codon).
      • Efficient translation of the leader sequence depends upon the concentration of charged tRNA's for the appropriate amino acids coded for by the mRNA leader sequence.
    • The leader sequence has the following characteristics:
    1. It contains a ribosome binding site and an AUG start codon necessary for the initiation of translation.
    2. The corresponding amino acid sequence coded for by the leader sequence contains several tryptophan residues.
    3. It contains several inverted repeat sequences such that the mRNA can adopt different alternative structures.
    4. One of these structures represents a rho-independent termination site.

    Screenshot (271).png

    Figure 2.7.4: High vs. Low tryptophan

    Under conditions of low tryptophan

    • A ribosome translating the nascent trp polycistronic mRNA stalls at the codons for tryptophan in the leader mRNA sequence.
      • This prevents segments 1 and 2 of the nascent mRNA from forming a potential stem-loop structure, and frees up segment 2 to form a stem loop structure with segment 3.
      • In this case, nascent transcription can proceed through, transcribing the entire trp operon.

    Under conditions of high tryptophan

    • A ribosome translating the nascent trp polycistronic mRNA does not stall at segment 1 (the region with codons coding for tryptophan).
      • In this case the stem-loop structure between 1 and 2 is not prevented from forming, and thus the stem-loop structure 3-4 can form.
      • This stem loop structure is essentially a rho-independent transcription termination structure.
      • The RNA polymerase transcribing the nascent message will terminate.

    Rho-dependent transcription termination

    • About half the termination sites in E. coli require an accessory protein called the rho factor.
    • Many rho-dependent sites have been characterized from E. coli and no obvious sequence similarity is present.
    • Rho associates with the nascent RNA transcript and this interaction activates an ATPase activity which appears to allow rho to translocate along the mRNA in the 3' direction.
      • It may be that if the RNA polymerase pauses, rho can catch up and cause termination (i.e. bump RNA polymerase off the DNA template?).
      • Pausing of RNA polymerase is thought to be an important mechanism of rho-dependent transcription termination.