Transcription (bacteria)

Key differences between DNA and RNA

DNA has deoxyribose; RNA has ribose. DNA has thymine; RNA has uracil. DNA is double stranded, RNA is usually single stranded

Why does DNA use thymine instead of uracil? See Peter tute 1 notes for more detail

• Spontaneous deamination of cytosine occurs when the amine group is attacked by water

• This forms uracil - if DNA used uracil, these deamination events would be difficult to recognise and repair

• Thymine has greater resistance to photochemical mutation (even though thymine is metabolically more costly to make)

• Instability in RNA is less critical as RNA is comparatively short-lied so any potential errors don’t lead to lasting damage

Transcription unit

Region of DNA that is copied (sometimes equivalent to a gene)

What is needed for the beginning and ending of transcription

Promoter sequences, terminator sequences

Components that RNA polymerase needs for transcription

• Double stranded DNA template (but no primer)

• Nucleoside triphosphates (adenosine TP, guanosine TP, cytidine TP, uridine TP) - the high energy phosphate bonds deliver the energy for RNA synthesis

Nucleobase vs nucleoside (e.g. A)

Adenine is the nucleobase, adenosine is the nucleoside

Where does the unwinding of DNA (forming the transcription bubble) and the synthesis of RNA occur

All within the RNA polymerase enzyme (approx 12-14 nucleotides)

Properties of RNA polymerase

• Unwinds a short stretch of DNA

• Only copies the template strand

• Incoming nucleotide must obey copying rules

• Incoming nucleotide joined to the 3’ OH of growing RNA, so RNA is synthesised in 5’ to 3’ direction

Why do polymerases always synthesise DNA/RNA in the 5’ to 3’ direction?

• The OH group of the growing strand carries out a nucleophilic attack on the incoming phosphate of the incoming nucleoside triphosphate

• They hydrolysis of the phosphate bond provides the energy for polymerisation

• If the incorrect nucleotide is added, the nucleotide is removed, which leaves the OH group again ready

• If it was the other way round, and nucleotides were added to the 5’ end, the energy source would be on end of the growing strand, so if a mistake was incorporated and the incorrect nucleotide was removed, the phosphate groups would no longer be on the terminal nucleotide in the chain

• This means that the energy to form the bonds would not be available, so the chain would terminate

• This means that proofreading would not be possible, so many more mistakes would be incorporated

• Triphosphates are also unstable, so the energy source would not only be lost in proofreading - it could happen in other circumstances as well

Formation of the transcription bubble

DNA is locally unwound for 12-14 base pairs. The DNA is kinked and pulled into the active site of the RNA polymerase.

mRNA in bacteria

Often polycistronic - contains multiple, tandem genes

Footprint

Area of a DNA sequence that cannot be hydrolysed by nucleases, since it is bound to RNA synthase, which protects it

How were key promoter elements in bacteria discovered

Through comparative genomics - comparing the genetically conserved sequences in the regions upstream of the genes

Where are the key elements in bacterial promoters

At the -10 (Pribnow box, rich in A-T pairing) and -35 positions (minus means upstream, it’s measured relative to the start of the gene)

How are the promoter regions linked to transcription

The sigma factor of E coli RNA polymerase recognises the promoter sequences, by making interactions with the DNA

What separates the DNA strands

The binding of the sigma factor to the -10 promotor region (Pribnow box) locally unwinds DNA: it causes bending of the DNA molecule, which leads to torsional stress, which bends and pulls the template strand into the active site. The non-template strand goes around the back of the sigma factor domain, and they come together outside of the enzyme. The Pribnow box is very rich in A-T pairs, which means that there is less hydrogen bonding there, so it is easier to separate the strands of DNA at that point.

Different sigma factors

• Sigma70 is the most common, responsible for most transcription in E coli including normal growth

• There are alternative, structurally distinct sigma factors that direct the transcription of specific genes in response to environmental signals

• These proteins are regulated by anti-Sigma proteins that bind sigma factors, preventing their access to the alpha2beta2 core polymerase

Structure of the DNA binding domain in the sigma70 factor

Helix-turn-helix motif - two helices held at a fixed angle - carboxy terminal helix is the recognition helix, and fits into the major groove of the DNA. Amino acid side chains recognise the specific DNA binding sequence.

Binding of DNA binding proteins to DNA

• Binding proteins contain structural motifs that can read DNA sequences - ~60 amino acid stretch involved

• The recognition sites are usually short

• The specific nucleotide sequence creates a pattern of structural features on the surface of the double helix

• Common pairings involving H bonds: Gln - A and Arg - G

When is the sigma factor released

After transcription begins

Two methods of transcription termination in bacteria

• GC-rich sequence, which folds into a stem-loop, followed by a U-rich tract (in the RNA). The protein NusA binds both the hairpin and the RNA polymerase in position, pausing transcription. The GC-rich sequence stabilises the stem loop, and the U-rich tract favours the dissociation of RNA from the DNA, since there are more hydrogen bonds in the GC-rich sequence. This pulls the RNA out of the RNA polymerase

• Rho-utilisation (rut) site - this is an RNA sequence that is bound by the ATP-dependent Rho helicase - a motor protein, which moves downstream down the RNA faster than the RNA polymerase, which reaches the RNA polymerase and continues to hydrolyse ATP to move down the RNA, which pulls the RNA out of the polymerase.

Main parts of a gene

Promoter, transcription unit, terminator

How are genes organised in a chromosome

As discrete units