transcription.docx

Prokaryotic genomes Not enclosed in the nucleus Has circular DNA + plasmids. Contains operons- which allows regulation of the expression of the genes contained within them. Gene operons are found clustered together in genomic island instead of being randomly dispersed around the circular genome....

Prokaryotic genomes Not enclosed in the nucleus Has circular DNA + plasmids. Contains operons- which allows regulation of the expression of the genes contained within them. Gene operons are found clustered together in genomic island instead of being randomly dispersed around the circular genome. Eukaryotic genomes Are enclosed within the nucleus. Arranged in linear chromosomes. Mostly non-coding for proteins (98%) Contains introns (26%)- sequences that are not translated. Around 59% of genome is repeated. Prokaryotic gene structure. Promoter region is important for RNA polymerase binding. Operator region binds other key regulatory factors that promotes or inhibits RNA polymerase access to gene promotor. Regulatory genes encode the factors that binds to operator and has its own promotor region, and its expression is controlled separately from the operon. Structural genes are all transcribed by the same promotor. Operon produces single RNA which is then translated to form different proteins. There are regions within the promotor where the sequence is very similar= consensus sequence. There are 3 regions. -35, -10 and +1 regions and the position of these 3 regions are important as it determines RNA polymerase binding. Eukaryotic gene structure Contains a promoter region that sits upstream of the transcription start site. Adjacent to the promoter region are proximal control elements- allows RNA polymerase to bind to the promoter. Also contains enhancers (distal control elements) which are 1000’s of bases upstream from promoter but as the DNA loops, these enhancers sit next to the transcription start site. Chromatin structure plays an important role in regulating eukaryotic gene expression. Gene contains exons (coding) and introns (non-coding) Also contains consensus sequences. Contains 3 regions upstream from the transcription start site. -25, -80 and -100. In prokaryotic, there is no nucleus separating transcription and translation. Once mRNA has been made, it can almost immediately be started to translate therefore transcription and translation can be happening to the same mRNA at the same time. In eukaryotes, there is a nuclear membrane which acts as a physical barrier between transcriptional and translational machinery. Transcription take place within the nucleus but translation happens on the ribosomes outside in the cytoplasm. Transcription is the process of RNA synthesis which requires DNA template. DNA template exists as 2 strands and are known as ‘sense strand’ and ‘antisense strand’. Sense strand carries the translatable code in the 5’ to 3’ region. Antisense strand is complementary to sense strand and acts as template for RNA. Prokaryotic transcription initiation. Prokaryotic RNA polymerase (RNAP) holoenzyme- multi subunit complex (5 subunits) that is formed when Core RNA polymerase binds with sigma factor. Without the sigma factor, the core RNA polymerase doesn’t recognise promoters but can still generate RNA. Sigma factors are transcription factors- proteins that regulate transcription and homologous to eukaryotic transcription factor TFIIB. There are Major and alternate sigma factors. Binds to RNAP and recognises specific -10 and -35 promoter sequences. Causes broad changes in gene expression usually in response to changes in environmental conditions. Different bacteria in different environments have different sigma factors eg E-coli has 7. There are additional molecules called anti-sigma factors that inhibit sigma factor function. During initiation of prokaryotic initiation: RNA polymerase holoenzyme binds to the promotor of a gene and begins to unwind the DNA. Holoenzyme binds to DNA and travel along the DNA molecule until it encounters the promotor sequences (-35 and -10). The complex stops and the holoenzyme starts to unwind the DNA double helix. Forms open complex and RNA synthesis begins. Sigma separates from the complex once a few phosphodiester bonds are formed between few nucleotides are produced. DNA is unwound by the RNA polymerase complex and phosphodiester bond formation in the new synthesised RNA molecule occurs within the active site. Newly synthesised RNA protrudes from RNA polymerase complex. Short region of RNA and DNA forms a heteroduplex. (important for termination step) RNA polymerase doesn’t simply bind to linear DNA, in fact the interaction involved both bending and wrapping of DNA around the RNA complex. The sigma factors contain different number of domains- recognise different elements within gene promoters= allow regulation. Eukaryotic transcription initiation Eukaryotic polymerase is much larger than prokaryotic one. It is made up to 12 subunits, but both have the same function. Eukaryotic RNA polymerase requires transcription factors to recognise promoters instead of sigma factors. Many other proteins are involved such as activators and mediators. Eukaryotes also contains 3 types of RNA polymerases. Each of them generates different RNA’s. TDIID complex binds to TATA box within the promoter sequence TFIIA binds to TDIID to stabilise the protomer initiation complex. TFIIB binds to B responsive elements that sit up and downstream of TATA box. RNA polymerase II/TFIIF complex binds. TFIIE and TFIIH binds, and transcription starts. Forms open promoter complex and requires ATP hydrolysis. All these TFIID and TFIIE etc are transcription factors subunits within the RNA polymerase complex and some of them are multi subunit complexes. Chromatin remodelling complexes actively remodel chromatin by changing the interaction between DNA and histone proteins. Allows nucleosome to be repositioned within the chromatin structure= allows access to promoter regions to allow transcription initiation to begin. Mediators- multi subunit complex of proteins and is regulated by kinases. Function is to mediate signals from transcription factors that are bound at enchanter region to transcription machinery. After the first few phosphodiester bonds are formed in the new RNA molecule, the RNA polymerase must be able to escape from promoter region. In prokaryotic, it is done when the sigma factor releases. TFIIH possess kinase activity therefore phosphorylates serine 5 of repeated sequence on carboxyl terminal of Rpb1 subunit of RNA polymerase complex (one of the subunits of RNA polymerase complex). Phosphorylation leads to recruitment of capping enzyme- the 5’ end of nascent transcript is capped. 5’ cap is important as it protects RNA from degradation and also promotes nuclear export for translation. Elongation step of transcription for eukaryotes During transcriptional elongation, a transient heteroduplex of DNA and RNA (important for termination) UTP is used instead of TTP (nucleotides). Transcriptional termination for prokaryotes Rho-independent transcriptional termination. ATP not required. Rho-dependent transcriptional termination Rho is a protein factor = RNA-binding ATP-dependent helicase Uses ATP to move along the RNA molecule. Rho binds to a sequence called Rho utilisation site (rut) on RNA transcript. Rut. Site is around 8- nucleotides long and rich in cytosine and poor in guanine. RNA polymerase pauses. Rho moves along the transcript towards the paused RNA polymerase via using energy from ATP hydrolysis. Rho then reaches DNA-RNA heteroduplex and helicase desiccates RNA polymerase complex causing termination. Eukaryotic transcriptional termination Triggered by polyadenylation signal 5’ AAUAAA 3’ on the RNA. RNA is cleaved 10-30 nucleotides downstream by the polyadenylation complex. RNA polymerase falls off template DNA. RNA processing In eukaryotic: RNA capping occurs during transcription. Stabilises transcript. RNA triphosphatase removes gamma-phosphate of the 5’ ribonucleotide. mRNA guanylyl transferase links GMP 5’ to 5’ to the beta-phosphate of the 5’ ribonucleotide. Methyl transferase methylates N7 of the GMP using S-adenosylmethionine. 2’ hydroxyls can also be methylated. Don’t need to know the process above, just know that different enzymes are used and the end result of capping is stabilisation of transcript. RNA splicing Occurs during transcription. Generates mature mRNA from precursor mRNA. Introns and removed and exons are put together. Mediated by spliceosomes which contains snRNP’s. Needs to be very accurate. Mature RNA is then exported from nucleus and different species of RNA with be associated with different chaperone proteins which guide RNA from nucleus to final destination.