Transcription Processes Notes

EXPRESSING GENES IN THE GENOME

RNA TRANSCRIPTION

For eukaryotic cell to make new protein a number of RNAs need to be transcribed in addition to the one for the protein itself (mRNA). mRNA is transcribed by RNA polymerase 2 and is translated into proteins

-these are transcribed by different isoforms of RNA polymerase- these are not translated into proteins but are catalytically active in their RNA form

-ribosomal RNA (rRNA-forms the core of ribosomes, catalyse protein synthesis)- RNA polymerase 1

-small nuclear RNA, miRNAs (regulate gene expression), mRNAs- RNA polymerase 2

-transfer RNA (tRNA- serves as adaptors between mRNA and amino acids)- RNA polymerase 3

-RNA polymerase are able to start transcription from de novo and don’t require a primer.

-first step in gene expression is transcription of the gene into a pre-mRNA. There are three stages: initiation, elongation and termination

GENE to pre-MRNA

Initiation

-first there is the opening of chromatin. Chromatin provides an additional level of gene regulation in eukaryotes.

-Heterochromatin- the nucleosomes are tightly packed and associated with additional heterochromatin proteins that bind to modified histone tails. DNA is inaccessible to RNA polymerase and prevents expression of genes.tightly.- constitutive heterochromatin: centromere/ telomere

Euchromatin- nucleosomes are more loosely packed, promoter and activator sequcences accesible. This exposes regions that are accessible to regulatory proteins. Increase in acetylation increases the exposure- active genes

Regulation of transcription at the level of DNA, nucleosomes, chromatin

- methylation on cysTeine or adenine switches of transcription,

-nucleosomes loosely packed- switches on transcription- there are chromatin remodelling complexes that bind in order to make the DNA more acessible

-Histone tails methylated-off Histone tails acetylated which loosens the grip

- then binding of the TATA box binding protein (TBP) subunit of transcription factor 2 D binds to the TATA sequence on the promoter region by recognising BRE sequences. The TATA box is located 25 base pairs upstream of start point

and TATA box binding protein factors (TAF, collectively known as TF11D) to the TATA box

-this causes the recruitment of other transcription factors for RNA polymerase 2- TF2 A, TF2 B (recognises BRE elements in the promoter), TF 2F

-This is followed by RNA polymerase 2 itself and finally TF 2E, TF 2H. The subunits of transcription factor H has kinase activity that phosphorylates C terminal of domain of polymerase 2 and this triggers transcription initiation. Phosphorylation of the C terminal of RNA polymerase II allows it to dissociate from other proteins and allows new set of proteins to associate with the RNA Polymerase tail. Processing proteins hop from the polymerase tail onto nascent RNA molecule to begin processing as it emerges from RNA polymerase

-The TFIIH also has helicase activity- on completion of its assembly, the initiation complex (basal transcription apparatus) unwinds a short stretch of double helix to reveal single stranded DNA that it will transcribe and interacts with transcription factors which regulates the rate of transcription

-promoters are upstream to the starting point and have a characteristic set of short conserved sequences which is recognised by basal transcription factors. Further upstream to the promoter region is the enhancer sequences that stimulate initiation. The proteins that bind to the enhancer sequence interact with proteins bound at the promoter elements through intermediates called coactivators

-REGULATION OF TRANSCRIPTION: the rate of transcription initiation by basal transcription apparatus is slow in the absence of enhancing transcription factors. Activators bind to a site distal to the promoter region, which activates coactivators which then activates the formation of the basal apparaturs. The additional factors ensure timely, controlled expression in response to tissue specific and proliferation cues.

Elongation

-RNA polymerase selects the correct ribonucleotide triphosphate that is complentary to the template strand and catalyses the formation of the phosphodiester bond

-the RNA polymerase does not require a primer and is synthesised in the 5’ to 3’ direction. If the lower strand is the template the RNA polymerase moves from left to right whereas if the upper strand was the template the RNA polymerase moves from right to left.

-Normal base pairing rules apply except uracil is base paired with adenine when it occurs in the template.

-the process is repeated many times and the enzyme moves unidirectionally away from the promoter region along the DNA template

-single enzyme transcribes a complete RNA. The RNA is different to DNA in two ways: ribonucleotide (ribose) and contains base uracil

-RNA polymerase doesn’t have any proof reading mechanisms- lower level of fidelity is less important as RNA is not inherited, also many copies of RNA are made so a few mistakes are unlikely to affect overall levels of protein synthesis.

Termination- signal lies within the newly formed RNA and not within the DNA

-As RNA polymerases are so processive- termination signals are needed to indicate when RNA transcription should stop.

-simplest stop signal is the palindromic GC region immediately followed by a T rich region

-the palindromic GC region forms a hairpin structure in the RNA due to base pairing. The T rich region causes an oligo U sequence after the hair pin and this destabilises the weak association between the mRNA and the DNA template- this leads to the dissociation of both the mRNA and RNA polymerase 2 from the DNA.

-DNA reforms the double helix

-there are also proteins that assist in terminating RNA transcription- one protein discovered in prokaryotes are rho protein which binds to C rich, G poor regions of the mRNA and scans along the RNA towards RNA polymerase in...

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