MBB222 - Lecture 26: prokaryotic transcription

what is the central dogma of molecular biology

a one way flow of genetic information → all cells express their genetic information in this way

what is the flow of molecular biology

DNA [ can also replicate ] — (transcription) → RNA —(translation) → protein

what is gene regulation

when different cells make different proteins at different times and in different amounts

why does gene regulation occur

occurs since genes have

• unique jobs

• change as they grow

• respond their environments

why does RNA base pair with DNA during transcription

• in order to copy genetic information correctly

• guides the building of the RNA strand

• ensure the correct code is passed on

why can/does RNA base pair with itself

• achieve thermodynamic stability

• protect its chemical groups

• to acquire biological function

why can short complementary strands of RNA form conventional and unconventional base pairing

• chemically flexible

• single stranded

• has multiple hydrogen bonding faces

• flexible spatial geometry

factors allow short complementary strands of RNA to form conventional/unconventional base pairing

for bacteria why does concentration of a given mRNA dictate the production of the protein it encodes

• Rnase enzymes quickly degrade mRNA → cell only makes proteins while that specific mRNA is present

• more mRNA copies - more chance for ribosomes to bind and make the protein

what 2 factors does mRNA concentration depend on

• rate of mRNA synthesis

• rate of mRNA degradation

what are the 3 elements of gene control in prokaryotes - which one is the most important

• transcription (most important)

• RNA turnover / nuclease degradation

• transcription termination

what is transcription initiation

the first phase of gene transcription where RNA polymerase binds to a gene’s promoter → DNA unzips → synthesis of RNA chain begins

what is RNA turnover

the continuous process of creating / breaking down RNA molecules inside the cell and is driven by rates of synthesis / degradation

what is transcription termination

the first step of gene expression → cell copies a gene’s DNA sequence into an RNA molecule

why is transcription the most important out of the 3 elements of gene control in prokaryotes

it decides whether a gene is copied at all → saves the cell enery if a gene doesn’t need to be copied

what is the differenc between coding strand and template strand

coding strand

• does not directly make the RNA transcript

• runs in the 5’ to 3’ direction

• is not used by RNA polymerase

template strand

• acts as the direct blueprint for building the new mRNA molecule during transcription

• runs in the 3’ - 5’ direction

• is used by RNA polymerase

what are the +1 / -10 / -35 regions in prokaryotes

are reference points and specific DNA sequences in bacterial promotes that direct RNA polymerase where and when to begin make RNA

what is the +1 region

the start site where RNA synthesis actually begins → first nucleotide of the DNA template is copied into RNA here

what is the -10 region

also known as pribnow box (TATA box)

• located about 10 base pair upstream (to the left of the start site → +1)

• area/region is rich in A-T base pairs

• -10 region facilitates unwind of the DNA template → A-T bonds are weak and melt first

what is the -35 region

• located about 35 base pairs upstream

• sequence acts a recognition region → sequence TTGACA is recognized and bound by the sigma factor ( a protein ) → helps in positioning RNA polymerase correctly

what is the UP element

• an A-T rich DNA sequence found upstream of the -35 region

• drastically boosts RNA synthesis by binding the RNA polymerase alpha subunit

what is the sigma factor

• helps RNA polymerase by guiding it to promoter DNA sequence

• locate specific gene start sites

• locks onto promoter DNA sequence

• opens the double helix

• forms the holoenzyme → combines with the core RNA polymerase to create a complete and functional unit that can recognize a specific DNA sequence

  • directs the enzyme to precise DNA sites / promoters (-10 / -35 regions)

  • reduces random binding by reducing the enzymes tendency to weakly / randomly stick to non target DNA

how is DNase I footprinting used to map protein binding sites

• by labelling a DNA fragment → binding a protein to it → cutting the DNA with DNase I enzyme

• bound proteins protect its specific sites from being cut

• leaves a blank gap or “footprint” on a size separation gel

what are the steps of transcription

• RNA polymerase binds nonspecifically to DNA

• shifts to high affinity binding when it encounters a promoter sequence (-10 / -35 region)

• closed complex is formed at the gene promoter

• DNA is partially unwound at the initiation site of transcription

• open complex is formed at the gene promoter

• RNA synthesis is initiated in a primer independent reaction

• sigma factor is released

• elongation occurs until termination

what is closed complex

the initial stable binding state where the RNA polymerase holoenzyme attaches to a double stranded promoter region → DNA remains completely base paired → start site has not melted

what is the oepn complex

functional stage where RNA polymerase unwinds about 15 base pairs of double stranded DNA around the transcription start site (+1) → exposes the template strand to begin RNA synthesis

what are the 3 main steps of transcription

initiation → beginning where RNA polymerase binds to the promoter

elongation → addition of nucleotides to mRNA strands

termination → end of transcription when RNA polymerase reaches a stop sequence

why does overwinding and under-winding occur during transcription

as RNA polymerase moves along the DNA it forces the strands to rotate

ahead of the polymerase (5’ direction) → unwinding of DNA leads to positive supercoiling

behind of the polymerase (3’ direction) → DNA strand re-anneal and under-wind leading to negative supercoiling

what is rho-dependent termination

• rho factor attaches to a specific spot on the new RNA strand (rut site)

• uses ATP energy to move along the RNA strand → catches up to the RNA polymerase

• once RNA polymerase pauses at a stop sequence → Rho catches up and unwinds the hybrid RNA/DNA bond (breaks bond with ATP) → releases the new RNA

what are the two ways termination can occur

• rho dependent termination

• rho independent termination

what is rho independent termination

• RNA polymerase copies a DNA section with reverse repeating letters → causes the new RNA to fold back onto itself → forms a stable hairpin shape

• hairpin structure causes RNA polymerase to slow down / stalls it

• RNA has a string of uracil bases → pairs weakly with the adenine in DNA template (weakens the hydrogen bonding between the hybrid DNA/RNA sequence) → causes the whole complex to break apart → releases the RNA