Functional organization of a eukaryotic gene
- Regulation of gene expression
- Promoter
- Site where RNA polymerase II and multiple other transcription factors bind to DNA upstream from gene locus (AT-rich upstream sequence with TATA and CAAT boxes).
- Promoter mutation commonly results in dramatic ↓ in level of gene transcription.
- Enhancer
- DNA locus where regulatory proteins (“activators”) bind → increasing expression of a gene on the same chromosome.
- Enhancers and silencers may be located close to, far from, or even within (in an intron) the gene whose expression it regulates.
- Silencer
- DNA locus where regulatory proteins (“repressors”) bind → decreasing expression of a gene on the same chromosome.
- Promoter
- RNA polymerases
- Eukaryotes
- RNA polymerase I makes ribosomal RNA, the most common (rampant) type; transcribe the 45S pre-rRNA gene into a single template that is subsequently processed into mature 18S, 5.8S, and 28S rRNAs. Present only in nucleolus.
- RNA polymerase II makes:
- Messenger RNA (largest RNA, massive). mRNA is read 5′ to 3′and is translated.
- Small nuclear RNA
- Micro RNA
- RNA polymerase III makes
- 5S rRNA (component of 60S ribosomal subunit)
- tRNA (smallest RNA, tiny).
- No proofreading function, but can initiate chains. RNA polymerase II opens DNA at promoter site.
- I, II, and III are numbered in the same order that their products are used in protein synthesis: rRNA, mRNA, then tRNA.
- α-amanitin, found in Amanita phalloides (death cap mushrooms), inhibits RNA polymerase II. Causes severe hepatotoxicity if ingested.
- Actinomycin D inhibits RNA polymerase in both prokaryotes and eukaryotes.
- Prokaryotes
- 1 RNA polymerase (multisubunit complex) makes all 3 kinds of RNA.
- Rifampin inhibits DNA-dependent RNA polymerase in prokaryotes.
- Eukaryotes
- RNA processing (eukaryotes)
- Initial transcript is called heterogeneous nuclear RNA (hnRNA). hnRNA is then modified and becomes mRNA.
- The following processes occur in the nucleus:
- Capping of 5′ end (addition of 7-methylguanosine cap)
- Polyadenylation of 3′ end (≈ 200 A’s)
- Poly-A polymerase does not require a template.
- AAUAAA = polyadenylation signal.
- Splicing out of introns from pre-mRNA by small nuclear RNA, removes introns containing GU at the 5′ splice site and AG at the 3′ splice site
- Capped, tailed, and spliced transcript is called mRNA.
- mRNA is transported out of the nucleus into the cytosol, where it is translated.
- mRNA quality control occurs at cytoplasmic processing bodies (P-bodies), which contain exonucleases, decapping enzymes, and microRNAs; mRNAs may be degraded or stored in P-bodies for future translation.
- Introns vs exons
- Exons contain the actual genetic information coding for protein.
- Introns are intervening noncoding segments of DNA. Different exons are frequently combined by alternative splicing to produce a larger number of unique proteins.
- Alternative splicing can produce a variety of protein products from a single hnRNA sequence (eg, transmembrane vs secreted Ig, tropomyosin variants in muscle, dopamine receptors in the brain).
- Introns are intervening sequences and stay in the nucleus, whereas exons exit and are expressed.
- Variants in which splicing occurs abnormally are implicated in oncogenesis and many genetic disorders (eg, β-thalassemia, Gaucher disease, Tay-Sachs disease, Marfan syndrome).
- RNA interference
- Short (20-30 base pair) non-coding RNA sequence induce post-transcription gene silencing.
- Silencing RNA: Small interfering (siRNA) and microRNA (miRNA).
- The human genome encodes >1000 miRNA genes, each one capable of repressing hundreds of target genes. Altered expression of even a few miRNA genes can lead to cellular dysregulation including hematologic and solid malignancies (by silencing an mRNA from a tumor suppressor gene.
- In addition, synthetic siRNA sequences can be introduced into specific pathogenic genes (eg c-myc oncogene) and are being explored as possible therapeutic agents.
- After being transcribed, miRNA is processed in the nucleus → forms double-stranded precursor → exported to cytoplasm → cleaved into a short RNA helix by a ribonuclease protein called dicer → strands separated & incorporated into RNA-induced silencing complex (RISC). → binds complementary sequences to miRNA found on target mRNAs →
- mRNA degradation (exact match)
- ribosome and transcription factor do not bind, leading to translation repression (partial match)