30 Gene expression: Transcription

Transcription

1 30 Gene expression: Transcription Gene structure. o Exons coding region of DNA. o Introns non-coding region of DNA. o Introns are interspersed between exons of a single gene. o Promoter region helps enzymes find the correct starting point for translation. Translation initiation occurs before the START codon in exon 1. o Terminator region includes regulatory sequences that are important but do not contribute to the protein. Translation finishes after the STOP codon in the final exon. o Un-translated regions (UTR) (blue) transcribed sequences that are not translated. 5 UTR and 3 UTR are important but not used to encode for amino acids. DNA strands. o DNA template strand. Strand used to generate complementary mrna sequence. Either DNA strand can act as a template strand. Direction of gene is always from 5 to 3 on the coding strand. But the direction of the gene is 3 to 5 on the template strand. Both strands do not encode the same gene. o DNA coding strand. Strand that is similar to mrna except that U is replaced by T. Not used in transcription. Contains the order of the amino acid sequence. mrna direction. o RNA transcription always occurs in a 5 to 3 direction. o Reverse complement mrna is always complementary to DNA template strand. Prokaryotes. o All genes are transcribed by a single RNA polymerase. o Made of 5 subunits (core - α 2 ββ ω) with a detachable sigma (σ) factor (holoenzyme). Core enzyme is required for polymerisation activities. Holoenzyme is required for binding to the promoter region (correct initiation of transcription).

2 Prokaryotic promotor binding (step 1 of transcription). o Sigma factor binds to the promoter region to determine where RNA synthesis should begin. Forms the closed complex. o RNA polymerase switches to an open complex. It melts the double-stranded DNA to form a transcription bubble. o Once the core enzyme is bound, the sigma factor dissociates. o σ 70 is the primary sigma factor, but there are other sigma factors for different purposes. o Function of each subunit. Alpha determine the DNA to be transcribed. Beta catalyse polymerisation. Beta bind and open DNA. Omega unknown Sigma recognise initiation sites (promoter regions). o Consensus sequences. Highly conserved sequences in the promoter region that help the sigma factor find the start of genes. Mutations in these sequences can result in the gene not being expressed. -35 and -10 (bp from start of transcription) regions (separated by bp) in the promoter for E. coli. TATA box = -10 region, sequence happens to be TAT and generally followed by AAT. Eukaryotic promotor binding (step 1 of transcription). o Eukaryotes have 3 RNA polymerases used for transcription. RNA polymerase I rrna (5.8S, 18S and 28S). RNA polymerase II mrna, some snrna. RNA polymerase III trna, rrna (5S), some snrna. o Promoters contain sequences that determine the specificity of the type of RNA pol binding. o Most promoters contain: Upstream regulatory elements. TATA box. Transcriptional start site. o General transcription factors. Necessary for the initiation of transcription bind promoter and facilitate RNA polymerase II binding. Undergo sequential binding with polymerase. TFIID binds to the TATA box. TFIIA and TFIIB subsequently bind. The complex is then bound by RNA polymerase, on which TFIIF is already attached. A pre-initiation complex is formed by the binding of TFIIE and TFIIH. TFIIH has ATPase and is responsible for unwinding the DNA helix and separating the two strands. Following ATP dependent phosphorylation, TFIIH forms the transcription bubble and RNA polymerase can now initiate transcription (without transcription factors).

3 o Specific transcription factors. Two types: activator and repressor proteins. Activator proteins bind to enhancer regions further upstream either proximal (close by) or distal (many bp away). Whether the STF are present or not determines if a given cell will initiate transcription or not. They cause the DNA to fold as the specific transcription factor binds to the initiation complex via mediators and co-activators. This interaction increases the rate of transcription. When a repressor protein binds to a silencer sequence which is adjacent to or overlapping an enhancer sequence, the activator protein cannot bind to the DNA. o These lead to high regulation of eukaryotic transcription, and thus is key to differential gene expression. Genetic elements that regulate transcription. o Tissue-specific transcription factors. o Repressors present in some regions and absent in others. Elongation (step 2 of transcription). o RNA polymerase breaks interactions with transcription factors and escapes the promoter region to start elongation. o RNA polymerase moves along the DNA template strand and adds bases in the 5 to 3 direction of the growing RNA strand. o Bases are complementary to the DNA template. o RNA polymerase binds to ~30 DNA bp at a given time. ~14 bp are unbound by RNA (in a transcription bubble). ~12 bp are bound as a RNA-DNA hybrid region. o There is progressive proof-reading, so it is possible to back up and correct mismatches. o Multiple RNAs can be transcribed simultaneously. Termination (step 3 of transcription). o Prokaryote termination - simple. RNA polymerase encounters chain termination sequence with high G-C content followed by at least 4 Us. Resulting RNA transcript is self-complementary and causes a hairpin to form with a stem and loop structure. RNA and RNA polymerase dissociates from the DNA. o Prokaryote termination rho dependent. DNA template contains a signalling sequence that is made of inverted repeats and is 40 bp long. The mrna sequence has a transcript of this sequence that is called the rho utilisation site (rut). Rho is an ATP-dependent helicase that binds to the rho utilisation site and moves along the RNA (requires energy). The terminator sequence in the DNA template causes the RNA polymerase to slow down. When the rho protein catches up to the RNA polymerase, it initiates termination of the RNA polymerase.

4 o Eukaryotic termination. Different for each RNA polymerase. RNA polymerase II. Passes the sequence 5 AAUAAA3. The cleavage and polyadenylation specificity factor (CPSF) binds to the sequence. A number of other factors including a cleavage stimulating factor and cleavage factor proteins also bind to form a complex. This complex causes the mrna to cleave. RNA processing in eukaryotes. o RNA is still in the nucleus. o 5 capping. Addition of a 7-methyl guanosine cap. Caps protect the growing RNA from degradation by nucleases. Recognised by translation machinery. o 3 polyadenylation. Facilitated by poly(a) polymerase. Addition of up to 200 adenine bases in the form of a Poly(A) tail. Enhances mrna stability and regulates transport to cytoplasm. o RNA splicing. Removal of introns. Primary transcript is spliced. Exons are joined up to make the final transcript. Small nuclear RNAs (snrna) joined with proteins form small nuclear ribonucleoproteins (snrnp). snrnps recognise the boundaries between introns and exons, in which they can recognise a number of different sequences. snrnps associate to the start and end of an intron that needs to be spliced out. They then interact with each other to form a spliceosome, causing the intron to form a loop. The spliceosome then cuts at the splice site and ligates the exons together. The intron that has been cut out is degraded in the nucleus.

5 o Alternative RNA splicing. Can result in different proteins from the same primary RNA transcript, which may be more suited to the cellular needs in different tissues/organs. Types: Exon skipping cutting out an exon by including it in the loop. Mutually exclusive exons integrate either one of two middle exons. Alternative 5 donor sites Can begin splicing in the middle of an exon, removing the latter part of the exon. Alternative 3 acceptor sites Can end splicing in the middle of an exon, removing the previous part of the exon. Drosophila sex determination. o Homodimers that result from the two X chromosomes in the female are transcription factors for the gene that produces the Sxl (sex-lethal) protein. o It binds to the promotor in the Sxl gene in females, and so the Sxl protein is made only in females. o The Sxl protein is important in coordinating the splicing in the next gene in the sequence, which is the Transformer protein gene (Tra). o There is a functional Tra protein in females, but not males. o The Tra protein promotes the use of an alternate splice site to skip a particular exon that codes for the male DSX (DSX-M) protein. o The result is a DSX-F protein in females for female differentiation and a DSX-M protein in males for male differentiation.