Lecture 2 RNA Structure, Function and Transcription v1.pdf

Molecular Diagnostics Transcription Lecture 2 Transcription • DNA directed RNA synthesis from a DNA template by a DNA-dependent RNA polymerase • Eukaryotic cell – DNA to message is nuclear – protein synthesis is cytoplasmic • Transport mechanism is necessary • mRNA translocate from nucleus to cyt...

Molecular Diagnostics Transcription Lecture 2 Transcription • DNA directed RNA synthesis from a DNA template by a DNA-dependent RNA polymerase • Eukaryotic cell – DNA to message is nuclear – protein synthesis is cytoplasmic • Transport mechanism is necessary • mRNA translocate from nucleus to cytoplasm RNA differs from DNA 1. RNA has a sugar of ribose DNA has a sugar of deoxyribose 2. RNA contains uracil (U) DNA has thymine (T) 3. RNA molecule is singlestranded DNA is double-stranded Structure and Properties of RNA • Unbranched polymer of nucleotide 5’ monophosphates linked by 3’-5’ phosphodiester bonds • Contains ribose instead of deoxyribose • Uricil replaces thymidine • Generally single-stranded (ss) except in some viruses • Can fold upon itself to form intrastrand helical regions. The filded 3D structure of RNA, like the folded proteins, can have multiple biological functions. Examples of Secondary Structure of RNA The ability of RNA to form unique 3D structures that act similarly to proteins is one of the key contributor that functional RNA can be involved in a wide range of cellular processes. Different Genes for Different RNAs • • • • • • • Pre-RNA mRNA tRNA rRNA snRNA snoRNA miRNA Ribosomal RNA (rRNA) 80% Transfer RNA (tRNA)15% Messenger RNA (mRNA) 5% RNA Synthesis • Synthesized on a DNA template • RNA polymerase • Reaction RNA polymerase/Mg2+ DNA template NMPn + ribonucleoside triphosphate NMP n+1 + PPi RNA Synthesis is Catalyzed by RNA Polymerase • Prokaryotes – 1 polymerase / 6 subunits – a2, b, b’, s, w (alpha-2,beta,beta prime, sigma, omega) – Sigma (s) subunit recognizes the promotor region of DNA that precedes the structural gene – Omega subunit (w) binds to promotor region, may serves to control the efficiency of RNA synthesis. But the overall function of omega (w) subunit is not very clear. – Rifampin- inhibits b subunit of polymerase Steps in Prokaryotic Transcription • Initiation – 15-20bp bubble formed and only 1 strand is read • Elongation – Starts with a purine, then continues • Termination – Rho independent (intrinsic stem-loop) – Rho dependent Prokaryotic RNA Polymerase Steps in Prokaryotic RNA Synthesis Positive and Negative Supercoils Generated by RNA Polymerase Eukaryotic Transcription Enzymes • More complicated • Hypersensitive regions of DNA • Several eukaryotic polymerases – Polymerase I-nucleolus-synthesizes 45s rRNA – Polymerase II-nucleoplasm, synthesizes mRNA, and others • recognizes promotor sequence – Polymerase III-nucleus, synthesizes tRNA, 5sRNA, miRNA – Mitochondrial polymerase-synthesizes mitochondrial RNA Transcription Eukaryotic Promotor • B-thalasemmia involves a mutation of TATA before b-hemoglobin gene • Affects rate of transcription and rate of protein production Primary Transcript • The primary transcripts – ~ 80 percent of the total RNA in rapidly growing mammalian cells is rRNA – ~15 percent is tRNA – ~ 5% of the total RNA is protein-coding mRNA • The primary transcripts are extensively processed to yield the mature, functional forms of various types of RNAs Primary RNA Processing Specific enzymes recognize the pre-RNA sequence and make incisions to generate pre-tRNA, pre-mRNA and pre-rRNA mRNA Processing – Many enzymes involved – Splicing (spliceosome complex) • Removal of introns • Connect exons • Alternative splicing: selectively to remove introns – tissue specific protein expression( a-tropomyosin, smooth vs striated muscle) – 3’ poy-A tail: many adenosines added for mRNA stability – 5’ cap: 7-methylguanosine to 5’ end Introns and Exons in Pre-mRNA Alternative splicing generate variants of mRNA and proteins with different functions and/or tissue specifics. Types of Processing for mRNA • 5’ cap: 7-methylguanosine to 5’ end • 3’ poy-A tail: many adenosines for stability • Splicing • removal of introns • alternative splicing – tissue specific protein expression( atropomyosin, smooth vs striated muscle) mRNA Processing Summary Transcription RNA polymerase binds to promotor region (via specific s factor) Promotor has specific sequences (CAAT,GC,TATA) DNA coding strand = sense strand Non-coding Strand (Template) = anti sense strand Anti-sense is the one used in the 3’-5’ direction so RNA chain is synthesized 5’-3’ and makes a coding strand • Coding strand has same sequence as message (except U instead of T) • Mature mRNA made in two sub-cellular compartments • • • • • Enhancer regions in Eukaryotic promotors effect regulation of gene expression. • EUK genes have many regions upstream that have sites for additional elements to bind • Binding to these sites will increase the rate and frequency of transcription • Affect tissue specific expression • Enhancer (site of binding of regulatory proteins such as transcription factors and hormone receptors) • Suppressor regions (site of binding of regulatory proteins that stop transcription) • Steroid hormone receptors binding • Cell signaling factors (100-5000 bp upstream) Hormone Binding Transcription Factor enhancer promotor Structural gene Post Transcriptional Modifications to Eukaryotic mRNA • Prokaryotes vs Eukaryotes • Purpose of modifications- G-cap and A-tailing – Stability of mRNA – Determines protein variation from a single message (splicing) – Guide for the translocation of mature mRNA to cytoplasm Inhibitors of RNA Synthesis • Template binding (at transcription initiation complex to prevent elongation) – Actinomycin D • Polymerase binding – Rifampin (b subunit) • a Amanitin (mushroom toxin) – Inhibitors of euk. pol II and III • Inhibitors of reverse transcription – AZT (azidothymidine) – ddI (dideoxyinosine) Nucleoside Analogues Human Diseases Related to mRNA Synthesis • Thalassemia – A form of inherited autosomal recessive blood disorder characterized by abnormal formation of hemoglobin. – Also affect rate of globin synthesis – US carrier rate= 7%; Mediterranean = 20% a Thalassemias – production of the α globin chain is affected – 4 alleles, mostly deletions b-Thalassemias – – – – – – production of the b globin chain is affected promotor mutations splicing mutations capping and tailing mutations nonsense and frame shift mutations dysfunctional protein is destroyed