Lecture 11 – Bacterial Transcription PDF

Lecture 11 – Bacterial transcription Part 1 – The background Expression levels of genes vary Transcription: The DNA sequence of each gene is transcribed into RNA molecules. Translation: The RNA molecules are then translated into protein sequences. Gene A, the transcription produc...

Lecture 11 – Bacterial transcription Part 1 – The background Expression levels of genes vary Transcription: The DNA sequence of each gene is transcribed into RNA molecules. Translation: The RNA molecules are then translated into protein sequences. Gene A, the transcription produces larger amount of RNA, higher expression at the transcription level. It produces more amino acids, very efficiently, more copies of gene A Gene B, the transcription produces a smaller amount of RNA and less amino acids. Poor efficiency so less copy of gene B Gene expression can be regulated at the level of transcription or translation or both. Essential polarity and strand nomenclature  The DNA molecule has a 5' to 3' directionality, with the 5' end on the left and the 3' end on the right. During transcription, the template (or "antisense") strand of DNA is used to synthesize an RNA molecule. The resulting RNA molecule has the same 5' to 3' directionality as the template DNA strand. The non-template (or "sense") strand of DNA has the same sequence as the resulting RNA molecule, except that it uses thymine (T) instead of uracil (U). The template (or "antisense") strand is complementary to the sense strand. The RNA synthesis follows the same base-pairing rules as DNA, with one exception - uracil (U) is used instead of thymine (T). mRNA is called sense strand (non-template copy). Uracil not found in DNA Cytosine can undergo spontaneous deamination to produce uracil. Cytosine pairs with Guanine Uracil pairs with Adenine  You lose amino group from cytosine to make uracil where oxygen take place of amino group. DNA replication after deamination would replace a C-G base pair with U-A base pair, introducing mutations. In DNA, any U is removed by uracil-DNA glycosylase, generating a basic site, which is removed by DNA polymerases. 3 major classes of bacterial RNA Type of RNA Function mRNA messenger RNA encodes proteins rRNA ribosomal RNA constituents of ribosomes: role in protein synthesis tRNA transfer RNA adaptors between mRNA and amino acids: role in protein synthesis These 3 classes of RNA molecules are synthesized by a single RNA Polymerase in Escherichia coli. Bacterial transcription unit Bacterial transcription unit includes the promoter, protein coding sequence and terminator.  Transcription and translation occur simultaneously in bacteria, as they lack a nucleus. The unit can contain multiple genes organized as a polycistronic operon for coordinated expression. 1. 5’ promoter (control by operator): attracts and binds RNA polymerase. Expect an operator 2. Transcribed (protein coding) sequence(s): often multiple genes – polycistronic - as part of an operon 3. 3’ terminator: signals the stop point for transcription Bacterial RNA polymerase: a multi-subunit protein complex The bacterial core RNA polymerase is composed of subunits α, β, β', and ω in the ratio 2:1:1:1. Addition of a σ subunit converts the enzyme to holoenzyme Subunit Roles: The α subunits play a role in the assembly and stability of the enzyme complex. The β and β' subunits form the catalytic core of the enzyme and are responsible for the actual RNA synthesis. The ω subunit helps regulate the activity of the enzyme. Addition of a σ subunit converts the enzyme to holoenzyme - is capable of recognizing and binding to promoter sequences on the DNA to initiate transcription. Promoters look like Methods: 1. RNA polymerase holoenzyme binds to DNA in vitro 2. Nuclease added (enzyme), this enzyme will degrade the DNA except for the region that is protected by the bound RNA polymerase. 3. The protected region, where the RNA polymerase is bound, will remain intact and form the "footprint" on the DNA. This footprint can be visualized and analyzed to identify the promoter sequence. DNA footprinting DNA Footprinting Technique: 32P-end-labeled DNA o DNA sample is labeled with radioactive 32P at the ends DNase Treatment o Labeled DNA is treated with limited amount of DNase enzyme o Partially degrades DNA, creating a ladder of fragments Protein Binding o DNA-binding protein (e.g. transcription factor) is allowed to bind to DNA Protected Region o DNA region bound by protein is protected from DNase cleavage o Results in a "footprint" with missing DNA fragments Gel Electrophoresis  o DNA fragments are separated by gel electrophoresis o Radioactive pattern is visualized o Protected region is revealed What does bacterial promoter look like? Usually, two regions are protected by RNA polymerase: one is centred around -10 bp (-10 sequence) from the start of transcription and the other centred around -35 bp (-35 sequence) from the start of transcription (+1). Conserved elements: -35 sequence TTGACA -10 sequence TATAAT +1 is usually either A or G Strong promoter sequences are closer to the consensus sequences The asymmetry of the promoter sequences provide directionality  1. The -10, -35 and +1 consensus sequences are defined on the SENSE (NON- TEMPLATE, NON-TRANSCRIBED) strand 2. RNA is built in the 5’ → 3’ direction: new nucleotides are added at the 3’ end, using the ANTISENSE strand as a template (don’t forget the pairing rules: G with C, A with U) Part 2 – The mechanism 3 stages of transcription Initiation – RNA polymerase holoenzyme binds the promoter, opens the DNA double helix and starts to transcribe Elongation - The σ subunit disengages from the holoenzyme, and the core enzyme continues to make new RNA. Termination – RNA polymerase core enzyme dissociates from the DNA, and transcription halts Initiation (1) The core RNA polymerase binds DNA non-specifically and can slide A σ subunit binds to the core polymerase and directs the polymerase holoenzyme to a promoter, binding to the -10 and -35 regions Escherichia coli has multiple σ subunits: different σ factors recognize different promoters, so they provide specificity. Initiation (2) - There are multiple E. coli σ factors Initiation (3) - scrunching, abortive initiation and success 1. Polymerase Pulling DNA: The RNA polymerase pulls the downstream DNA towards itself, a process known as "scrunching." 2. Scrunching the DNA: This scrunching action creates tension and stress in the DNA. 3. Abortive Initiation: If the -10 region of the promoter is not successfully opened, the polymerase will continue to scrunch the DNA until this region is opened. This is known as "abortive initiation," where the polymerase attempts to initiate transcription but fails. 4. Successful Initiation: Once the -10 region is opened, the polymerase can convert the "CLOSED" promoter complex into an "OPEN" promoter complex. This step does not require the energy of ATP hydrolysis, unlike the action of DNA helicases. 5. Topoisomerases: The note mentions that topoisomerases can relieve the problems of supercoiling, which is a result of the scrunching and tension in the DNA. Initiation (4) 12 to 15bp are unwound, from within the –10 region to position +2 to +3: the transcription start site is exposed RNA RNA polymerase now makes an RNA copy from the template strand, using base pairing rules (G with C, A with U). Unlike DNA Pol, RNA polymerase does not require a primer After about 10 nucleotides of RNA synthesis, the σ factor is exposed and disengages. The RNA polymerase can now elongate the new RNA. Elongation: a transcription ‘bubble’ During elongation, RNA polymerase is highly processive. The RNA: DNA hybrid is ~8bp long. The unwound DNA bubble is ~17bp long. Rate of elongation: slow compared to DNA pol, about 50 nucleotides/sec Elongation: proofreading Termination of transcription in bacteria There are 2 mechanisms Rho (ρ)-independent: a terminator sequence in the RNA is recognized ρ-independent: requires ρ protein to break the RNA: DNA duplex in the transcription bubble  In both cases, the functioning signals are recognized not in the DNA template, but in the newly synthesized RNA.