Transcription in Bacteria General Terms

Promotor

Regulatory sequence that acts as the binding location of RNA polymerase to initiate transcription

Coding region

Region of a gene that codes for a gene product; the sequence that harbors the sequence for a protein

Termination Region

Region of the gene containing the transcription-terminating sequence; when polymerase reaches this region it stops transcription.

Transcription Start Site

Position on the gene that corresponds to the starting point of transcription and is identified as the +1 nucleotide

False; the promotor region is NOT transcribed

The promotor region is transcribed. T/F

One;

How many types of RNA polymerase do bacteria have?

Pentameric RNA polymerase core (~390 I’d)

What is the core enzyme of the bacterial rna polymerase

Core enzyme

component of RNA polymerase that carries out transcription but cannot recognize and bind to promotor region

Sigma subunit

A protein component of RNA polymerase that is responsible for recognizing the promoter region of DNA during the initiation of transcription.

Holoenzyme

Active form of a core enzyme

False; must bind with a sigma subunit to be able to recognize promotor region

T/F RNA polymerase can bind to promotor and begin transcription on its own

Consensus Sequence

A consensus sequence is a derived sequence representing the most common nucleotides at each position of a set of related sequences; it is used to identify binding sites for proteins, like transcription factors.

Different forms of sigma subunits induce distinct conformational changes to the RNApolymerase holoenzyme and to the recognition of distinct promoters

What is the importance of alternate sigma subunits?

Rifampicin

Inhibits RNA synthesis by preventing RNA polymerase from catalyzing the formation of the first phosphodiester bond on the RNA chain; A single mutation of RNA polymerase can change a bacterium from sensitive to resistant to this antibiotic

I) Initiation of transcript synthesis

II) Elongation of transcript

III) Termination of transcript

What are the three stages of bacterial transcription

Transcript initiation (step 1)

• RNA polymerase is a large molecule that binds to the -35 and -10 (pribnow) consensus sequences, occupying the space between and around the sites

• Holoenzyme (active form) makes an initial loose attachment to double strand promotor sequence and then tightly binds to it to form the closed promotor complex

Closed promotor complex

The closed promotor complex is formed when RNA polymerase tightly binds to the double-stranded promoter sequence, occupying the space around the -35 and -10 consensus sequences before transcription begins.

Transcription Intiation (step 2)

The bound holoenzyme unwinds approx. 18 bp of DNA around (downstream) the Pribnow consensus sequences to form the open promotor complex.

Transcription Intitiation (step 3)

• holoenzyme progresses downstream to initiate RNA synthesis at +1 nucleotide on the template strand (no coding/antisense) of DNA

• Holoenzyme remains intact until the first 8-10 RNA nucleotides have been joined; soon after, sigma subunits disassociates and core enzyme continues transcription

Transcription Elongation

• Core enzyme continues synthesizing the RNA at a rate of ~40 nucleotide s/sec

• Progression of core enzyme is accompanied by DNA unwinding ahead downstream (via helicase)

• Duplex closes following synthesis

Transcription Termination

Transcription Terminates when the core enzyme encounters a termination sequence.

• Termination sequences are located downstream of stop codons. They are transcribed but NOT translated

• Two mechanism of Transcription termination in bacteria:

  • Intrinsic & Rho-dependent

Intrinsic Termination

Depends on the occurrence of specialized repeat sequences (inverted repeats) in DNA which induce the formation in RNA of a secondary structure leading to transcription termination.

• The transcription of these inverted repeats produces mRNA with complementary segments; these complementary segments can fold into a short, double-stranded stem, ending with a single stranded loop/hairpin structure → followed by a poly-U sequence.

• The formation of the stem-loop structure followed immediately by a poly-U near the 3’ end of RNA, causes RNAP to slow down and destabilize.

• This instability induces RNA polymerase to release the transcript and separate from DNA

Polyadenine Sequence

A string of adenines on the template strand of DNA which begins at the 5’ end of the inverted repeats 2 region; characteristic of intrinsic termination sequences.

Rho-Dependent Termination

• Certain bacterial genes require the action of rho protein to bind to nascent mRNA and catalyze the separation of mRNA from RNA polymerase

• As RNA polymerase continues to elongate the mRNA in the 5’-3’ direction, rho protein attaches itself to the rut site and quickly towards the RNA polymerase

• RNA polymerase reaches and transcribes the termination sequences containing an inverted repeat sequence; a stem-loop forms in the mRNA, causing the RNAP to slow so rho protein can catch up

Rho ultilization site

A sequence on DNA, distinct from intrinsic termination sequences, that will be transcribed to the rut sequence.

Rut sequence

A sequence on RNA that will be recognized by the rho protein

Rho Protein

Terminates transcription by catalyzing the release of mRNA from RNAP and causing RNAP to drop off of DNA