Overview & transcription I

What is a gene (from a molecular biology perspective)?

A sequence of genomic DNA that encodes a single, functional RNA

What is included in the coding region of a eukaryotic genome?

exons = expressed segments

introns = intervening segments

promotor = ensure gene is transcribed at the correct time/place

How is speed prioritised in eukaryotes in transcription?

No nucleus

no introns

mRNA is translated as it is being transcribed

What direction does transcription occur in?

5’ to 3’ direction

What is required for mRNA synthesis in prokaryotic cells?

DNA templates to copy

ribonucleotides (e.g. ATP, GTP, CTP & UTP)

No need for a primer

Why is the error frequency much higher in transcription than DNA replication?

RNA polymerase has no proof-reading 3’-5’ exonuclease activity

What are the 2 forms of RNA polymerase and what are their subunits?

Core enzyme: alpha₂, beta, beta’ subunits

Holoenzyme: alpha₂, beta, beta’ & sigma subunits

At what sequence does RNA polymerase bind to begin transcription?

The promotor

How do core RNA polymerases recognise the promotor in bacteria?

The sigma factor binds to the core enzyme to form a holoenzyme

Sigma factor has a high affinity for a unique promotor & suppresses the random non-specific binding on the DNA.

Describe the steps of initiating RNA synthesis.

Holoenzyme binds to promotor but DNA strands stay closed

DNA strands open = open complex

Enzyme selects appropriate base for complementary RNA strand

Ribonucleotide incorporated and pyrophosphate eliminated

Moves on to the next nucleotide and repeats

What is the definition of promotor?

Sequence of DNA needed for the RNA polymerase to bind to the template and initiate RNA synthesis

How is the promotor assymetric?

Sequence read 5’ to 3’ on one strand is very different to the sequence read 5’ to 3’ on the other DNA strand

Why does it not matter that the promotor is assymetric?

RNA polymerase enzyme is asymmetric

Therefore there is only one orientation that the enzyme will bind to the DNA

DNA strands will lie in different sites on the enzyme

What are the two types of bacterial termination sites?

rho independent & rho dependent

What causes termination at the rho independent RNA site?

G-C rich hairpin followed by a run of approximately 6 uracils in the RNA

Pauses RNA polymerase and causes it to dissociate from the DNA

What causes termination in rho dependent sites?

Have the C-G hairpin that pauses the polymerase

Enzyme Rho binds the C-rich DNA and unwinds the RNA-DNA duplex whilst the polymerase is paused at the hairpin

How does a repressor protein work?

Binding site overlaps that of RNA polymerase

Blocks RNA synthesis when bound to DNA

How do positive regulators work?

Binds to specific DNA sequence

Enhances efficiency of RNA polymerase entry, binding & initiation of transcription

What is actinomycin D?

Molecule that blocks all RNA synthesis immediately

Binds tightly to DNA

Too toxic for clinical use but may be used for some cancers

What is rifamycin?

Antibiotic that blocks all bacterial RNA synthesis

Does not affect eukaryotic transcription therefore used in tuberculosis treatment.