Transcription in Prokaryotes

 RNA polymerase activity: some similarities, some differences to DNA polymerase activities

     Similarities:

• uses nucleoside 5'-triphosphates (NTPs) as precursors
• catalyzes phosphodiester bond between NTPs
• uses DNA as template
• base pairing determines correct base
• growth of nucleic acid chain only in 5' to 3' direction
• growing strand antiparallel to template strand

    Differences:

• uses ribonucleoside 5'-triphosphates instead of deoxyribonucleoside 5'-triphosphates (ATP, GTP, CTP, UTP)
• can initiate the start of a new strand de novo (no primer necessary) (remember the RNA polymerase "primase" in replication)
• a single strand of RNA is produced (only one strand of DNA used for RNA synthesis: the DNA template strand with complementary base sequence)
• only short stretches of DNA are transcribed
• Only one RNA polymerase to make all RNA, i.e., mRNA, rRNA, and tRNA.

E. coli RNA polymerase

• Very large protein complex, consists of five subunits (Fig 10.10).
• 2 identical alpha subunits and 1 each of beta, beta', and sigma.
•  The sigma subunit dissociates from the enzyme easily - leaves shortly following initiation, critical for recognition of start of gene.
•  holoenzyme - complete enzyme - all five subunits together but basic polymerization reaction possible without sigma subunit -
• core enzyme: 2 alpha, 1 beta and 1 beta'

• The base where transcription starts is numbered +1.
• Bases distal of the +1 base from the coding region  are referred to as upstream and are number as: -1, -2, -3  ...
• Those bases adjacent to the +1 base close to the coding region are referred to as downstream and are positively numbered.
• The regions of DNA that bind RNA polymerase are called promoters.
• The regions of DNA that bind regulatory proteins are called operators.

Consensus Binding Sites

• Alignment of several promoters helps identify common sequences that may be important in protein binding (Fig. 10.11a).
• A consensus sequence can be derived from the most conserved bases (Fig. 10.30).
• Mutations of bases a specific positions can verify the functional importance of specific bases (Fig. 10.11b).

Promoters

• Bacterial promoters have two consensus sequences centered at -10 and -35 (Fig. 10.11b).
• The RNA polymerase core uses the -10 sequence (TATAAT) to bind and orient where to begin transcription.
• The RNA polymerase sigma subunit uses the -35 (and -10 to some degree) sequence to bind and stabilize the polymerase complex.
• Different sigma subunits can direct the polymerase to specific genes.  Synthesis of new or unique sigma subunits can redirect the polymerase to transcribe a new set of genes (Table 10.1).
• The strength of a promoter is related to its identity to the consensus sequence (Fig. 10.11c).
• Not all genes have the -10 and -35 sequence in the promoter.

• Most operators are short inverted repeats (Fig. 10.12).
• Most DNA binding proteins have dimer or multimeric subunits containing alpha-helices that bind in the major groove of the operator DNA (Fig. 10.14).
• Negative regulator proteins bind such that they prevent RNA polymerase binding or movement (Fig. 10.9).
• Positive regulator proteins often interact with the RNA polymerase and stabilize it at poor promoters that do not closely match the consnsus sequence (Fig. 10.17).
• Enhancers can accelerate the conversion of closed complexes into open complexes (Fig. 10.19).
• Many bacterial responses are controlled by two-component regulatory systems (Fig. 10.19, Fig. 10.21).

Phases of transcription

    Specific binding of RNA polymerase:

• RNA polymerase can bind to double-stranded DNA nonspecifically with low affinity (low strength)
• The holoenzyme (with sigma subunit) binds specifically to both the -10 and -35 sequence with high affinity ---- resulting in a closed-promoter complex
• The holoenzyme unwinds about 15 bases in double-stranded DNA around the initiation site for transcription ---- resulting in an open-promoter complex

• Transcription starts by base pairing of two rNTPs that are joined (therefore, the very first ribonucleotide in a newly transcribed RNA retains the triphosphate)
• When approximately 10 rNTPs are jointed by phosphodiester bonds [resulting in the loss of 2 phosphate groups from each rNTP], the sigma subunit is released from the core enzyme

• The core enzyme leaves the promoter site
• The core enzyme travels along DNA template
• The DNA is unwound in front and is rewound behind so that approximately 17 base pairs are always unwound
• rNTPs are added to growing RNA via base pairing and phosphodiester bond formation

• The core enzyme encounters a termination signal
• RNA is released from DNA template and from the enzyme

RNA polymerase dissociates from DNA

Quiz