Promoters and the Initiation of Transcription

I. General Properties

• A promoter is the DNA sequence required for correct initiation of transcription

   

• Mutations in promoters affect the amount of product from a gene but do not affect the structure of the gene product.

II. Bacterial promoters

• Occur just 5' to and overlap the start site for transcription

   

• Binding site for E. coli RNA polymerase holoenzyme
  • Covers about 70 bp from about -50 to about +20.

   

• Consensus sequences in the E. coli promoter
  • -35 and -10 sequences

                         -35       16-19 bp      -10     +1

--------TTGACA-------------------TATAAT---CAT

 Recognition by  RNA polymerase       Allows binary complex to
                                                           convert from closed to open

• Conserved in all E. coli genes transcribed by holoenzyme with s70
• Genes for more than 100 sigma proteins have been sequenced
  • the proteins form a large family, the s70 family of sigma factors
  • one important exception is s54, which shows no homology to s70 proteins, and which interacts with core polymerase differently from members of the s70 family.
• members of the s70 family of sigma factors have 4 conserved regions (Figure adapted from Fig 7-3, Kornberg and Baker, DNA Replication, WH Freeman and Co, p 238):
  
  • Region 4:
    • a DNA binding activity that recognizes the -35 promoter motif
      • this is inferred from allele specific mutations that alter promoter recognition
  • Region 2
    • Subregion 2.3 and 2.4 form a DNA binding activity that recognizes the -10 promoter motif
      • this is inferred from
        • allele specific mutations that alter promoter recognition, and
        • from X-ray structure of this portion of s70 (Malhotra et al., 1996, Cell 87:127-136)
          • <> Acids (blue) - maybe contact backbone
          • <> Residues known to interact at -12 position (orange)
          • <> conserved hydrophobic core (yellow) - structural integrity of this domain?
          • <> clusters of aromatic amino acids that are solvent exposed (cyan) which may be used to intercalate between bases to stabilize single stranded DNA, thus contributing to melting the promoter region
            
          • <> all of the above
            
    • region 2 also interacts with core enzyme components
      • this part of s70 is conserved with the eukaryotic factor RAP30
      • rotate the previous view to look behind it, and identify atoms:
        • <> red - promoter melting
        • <> green - mutations that purturb RNAP binding
        • <> yellow - exposed hydrophobics, highly conserved, for RNAP binding?
        • <> blue - other exposed, highly conserved residues
        • <> all of these combined
  • Region 1
    • masks the DNA binding activities that are present in regions 2 and 4, which become unmasked when the sigma factor binds to the core RNA polymerase
      • in region 2 this may involve an acid loop, as described by Malhotra et al, 1996, Cell 87:127-136, and adapted here:
        
  
  • Nonspecific DNA Binding
    • a 245-amino acid region between 1 and 2 is in s70, but not many other sigma factors
      • this portion of s70 is believed to reduce the nonspecific binding activity of core, thus enhancing promoter specificity
      • in Bacillus subtilis, which lacks this region in its s43 protein, a second peptide called d21 serves this function.

   

• Promoter mutants
  • Tend to fall into or close to one of the -35 or -10 hexanucleotides
  • Affect the level of gene expression, not the structure of the gene product
  • Down promoter mutations: decrease the level of transcription. Tend to make the promoter sequence less like the consensus.
    • Down promoter mutations in the -35 sequence: decrease the rate of formation of the closed complex, indicating this is the sequence needed for initial recognition by the polymerase holoenzyme.
    • Down promoter mutations in the -10 sequence: decrease the rate of conversion from the closed to the open complex, again supporting the proposed role for this A+T rich hexanucleotide.
  • Up promoter mutations: increase the level of transcription. Tend to make the promoter sequence more like the consensus. 
  • The critical contact points between RNA polymerase and the promoter tend to be in or immediately upstream from the consensus -35 and -10 boxes. Thus the biochemical and genetic data all support the importance of these conserved sequences.

   

• Alternate s factors can control the expression of sets of genes
  • Alternative s factors make complexes with the core polymerase to direct the new holoenzyme to a particular set of promoters that differ in sequence from the general E. coli promoter sequence. Thus the polymerase can be directed to transcribe a new set of genes. This is one way to control gene expression.
  • Examples include s factors for heat-shock response (s32), nitrogen starvation (s54), and transcription of genes involved in chemotaxis and flagellar formation (s28). The s factors are named by their size in kDa.

   

  s factor	Gene	Use	-35	Separation bp	-10
  s70	rpoD	General	TTGACA	16 to 19	TATAAT
  s32 = sH	rpoH	Heat shock	CCCTTGAA	13 to 15	CCCGATNT
  s28 = sF	fliA	Flagella	CTAAA	15	GCCGATAA
  			-25		-12
  s54 = sN	rpoN	Nitrogen starvation	CTGGNA	6	TTGCA

   

• Complex interplays between sigma factors, anti-sigma factors, and two-component signal transduction provides facile regulation of developmental activities.

  For example, consider sporulation in Bacillus subtilis.

III Eukaryotic Promoters

• The overall shape of Prokaryotic and Eukaryotic RNAP are similar.
  

   

• General transcription factors: required for Pol to bind.
  • Factors are required for transcription from a minimal promoter that are not subunits of purified RNA polymerase
  • Factors are named TFIIx, where x = A, B, ... designating some chromatographic fraction that is required for in vitro transcription. TFII stands for transcription factors for RNA Pol II. They are TFIIIx for factors for RNA Pol III.
    • TFIID is a complex of many subunits. It includes the protein that binds specifically to the TATA box, called TATA binding protein = TBP, plus several TBP-associated factors, or TAFs.
    • TBP binds in the narrow groove (minor grove) of DNA at the TATA box, and bends the DNA.
    • It is not known if the same set of TAFs are in the TFIID for all promoters transcribed by RNA polymerase II, or if some are used only for certain types of promoters. TFIID is the only sequence-specific general transcription factor so far characterized, and it binds in the minor groove of the DNA. It is also used at TATA-less promoters, so the role of the sequence -specific binding is still under investigation (see tbp).