* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Fundamentals of Nucleic Acid Biochemistry: RNA
X-inactivation wikipedia , lookup
Alternative splicing wikipedia , lookup
Secreted frizzled-related protein 1 wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Genome evolution wikipedia , lookup
Community fingerprinting wikipedia , lookup
Transcription factor wikipedia , lookup
Histone acetylation and deacetylation wikipedia , lookup
Gene expression profiling wikipedia , lookup
List of types of proteins wikipedia , lookup
Molecular evolution wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
RNA interference wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Non-coding DNA wikipedia , lookup
Endogenous retrovirus wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Gene regulatory network wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Messenger RNA wikipedia , lookup
Polyadenylation wikipedia , lookup
RNA silencing wikipedia , lookup
RNA polymerase II holoenzyme wikipedia , lookup
Eukaryotic transcription wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Non-coding RNA wikipedia , lookup
Gene expression wikipedia , lookup
Epitranscriptome wikipedia , lookup
Fundamentals of Nucleic Acid Biochemistry: RNA Donna Sullivan, PhD Division of Infectious Diseases University of Mississippi Medical Center DNA AND RNA: BASIC STRUCTURE Repeating Nucleotide Subunits In DNA and RNA STRUCTURE OF RNA SUGAR Ribose Phosphate group Nitrogen containing base Adenine Guanine Cytosine Uracil THE NUCLEOTIDE: RNA OH O=P-O-5CH2 BASE OH O 4C H 1C H 3C OH H 2C 0H H Adenine Guanine Cytosine Uracil The Forms Of RNA: Not Just Another Helix Does not normally exist as a double helix, although it can under some conditions Can have secondary structure: Hairpins: pairing of bases within 5-10 nt Stem-loops: pairing of bases separated by >50 nt Can have tertiary structure: Pseudoknot, cloverleaf RNA Structures Structure Of RNA Structure of tRNA Structure of rRNA RNA Replication vs. DNA Replication RNA replication: Requires no priming Has many more initiation sites Is slower (50–100 b/sec vs. 1000 b/sec) Has lower fidelity Is more processive Transcription Transcription is the enzyme-dependent process of generating RNA from DNA. The process is catalyzed by a DNA-dependent RNA polymerase enzyme. Only “coding” segments of DNA (genes) are transcribed. Types of genes include structural genes (encode protein), transfer RNA (tRNA), and ribosomal RNA (rRNA). Transcription Transition from DNA to RNA Initiation: Gene recognition RNA polymerase enzyme and DNA form a stable complex at the gene promoter. Promoter: Specific DNA sequence that acts as a transcription start site. Synthesis of RNA proceeds using DNA as a template. Only one strand (coding strand) is transcribed, the other strand has structural function. Transcription factors are proteins that function in combination to recognize and regulate transcription of different genes. Termination signal RNA POLYMERASES Cellular RNA polymerase RNA pol I transcribes rRNA genes RNA pol II transcribes protein encoding genes RNA pol III transcribes tRNA genes Viral RNA polymerases Reverse transcriptase of retroviruses RNA dependent RNA polymerases of negative stranded viruses RNA Polymerase Enzymes (DNA-dependent RNA Polymerase) RNA Polymerase I transcribes most rRNA genes (RNA component of ribosomes). RNA Polymerase II transcribes structural genes that encode protein. RNA Polymerase III transcribes tRNA genes (for transfer RNAs). RNA Polymerase IV is the mitochondrial RNA polymerase enzyme. RNA TRANSCRIPTION General Organization of a Eukaryotic Gene Promoter/Enhancer Enhancer Region Regulatory Proteins Promoter Region Regulatory Proteins RNA TFIID Polymerase II TATA 200–2000 bp 100–200 bp 200–10,000 bp ~30 bp Gene Structure Upstream 5´ Downstream Exon 1 Promoter Intron 1 5´- UTR (Untranslated Region) Exon 2 Intron 2 Exon 3 Intron 3 Exon 4 3´ 3´- UTR (Untranslated Region) Polyadenylation Signal Nuclear Processing of RNA Chemical modification reactions (addition of the 5´ CAP) Splicing reactions (removal of intronic sequences) Polyadenylation (addition of the 3´ polyA tail) Processing of mRNA In eukaryotes, the mRNA molecule that is released after transcription is called precursor mRNA or pre-mRNA. It undergoes several changes before being exported out of the nucleus as mRNA. Processing of mRNA 5’ end is capped with a modified form of the G nucleotide known as the 5’ cap At the 3’ end, an enzyme adds a long series of A nucleotides referred to as a poly-A tail It serves to protect the mRNA from enzymes in the cytoplasm that may break it down The greater the length of the poly-a tail, the more stable the mRNA molecule RNA Transcription and Processing The process of RNA transcription results in the generation of a primary RNA transcript (hnRNA) that contains both exons (coding segments) and introns (noncoding segments). The noncoding sequences must be removed from the primary RNA transcript during RNA processing to generate a mature mRNA transcript that can be properly translated into a protein product. RNA Processing Capping of the 5´-end of mRNA is required for efficient translation of the transcript (special nucleotide structure). Polyadenylation at the 3´-end of mRNA is thought to contribute to mRNA stability (PolyA tail). Once processed, the mature mRNA exits the nucleus and enters the cytoplasm where translation takes place. RNA Processing Biogenesis of Mature mRNA Genomic DNA Boundary Transcriptional Promoter Exon 1 Intron 1 Exon 2 Intron 2 Exon 3 Gene Structure in Chromosomal DNA Transcription Initiator ATG Stop PolyA Codon Signal Primary RNA Transcript Posttranscriptional Processing Initiator ATG Mature mRNA Transcript Stop Codon PolyA Tail Genomic DNA Boundary RNA Processing Fundamentals of RNA Splicing Primary RNA Transcript Exon 1 Intron 1 Exon 2 …NNAG GUAAGU CAG GNNN… Exon 1 Exon 2 5´ Splice Junction 3´ Splice Junction RNA Splicing Exon 1 Exon 2 Mature RNA Transcript RNA Processing: Spliceosomes Ribonucleoproteins (snRNPs) function in RNA processing to remove intronic sequences from the primary RNA transcript (intron splicing). Alternative splicing allows for the generation of different mRNAs from the same primary RNA transcript by cutting and joining the RNA strand at different locations. snRNPs are composed of small RNA molecules and several protein molecules. Five subunits form the functional spliceosome. RNA Processing Alternative Splicing of RNA Normal Splice Site Exon 1 Alternative Splice Site Exon 2 RNA Splicing Normal Transcript Alternative Transcript Exon 3 Structural Genes Encode Proteins The majority of structural genes in the human genome are much larger than necessary to encode their protein product. Structural genes are composed of coding and noncoding segments of DNA. Structural Genes Encode Proteins The structure of a typical human gene includes informational sequences (coding segments termed exons) interrupted by noncoding segments of DNA (termed introns). The exon-intron–containing regions of genes are flanked by non transcribed segments of DNA that contribute to gene regulation. Factors That Control Gene Expression Include: Cis elements are DNA sequences. Trans elements are proteins. Control of Gene Expression Primary level of control is regulation of gene transcription activity. TATA box contained within the gene promoter provides binding sites for RNA polymerase. Enhancer sequences can be sited very far away from the gene promoter and provide for tissuespecific patterns of gene expression. Gene Enhancer Sequences 5´ Exon 1 Intron 1 Enhancer Promoter 5´-UTR 1. Enhancer sequences are usually sited a long distance from the transcriptional start site. 2. Enhancers maintain a tissue-specific or cellspecific level of gene expression. 3. The gene promoter contains TATA box upstream of transcription start site. Protein binding sequences in the ‘Promoter’ region Gene Regulation: Two types of regulation Negative regulation Substrate induction (lac operon): gene OFF unless substrate is present End product repression (trp operon): gene OFF if end product is present Common in bacteria Positive regulation Gene is OFF until a protein turns it ON Regulatory proteins turn gene ON Occurs in eukaryotes Negative Regulation Positive Regulation Lac Operon Operon Gene organization in bacteria in which several proteins are coded by one mRNA Allows all proteins to be controlled together Differences Between Prokaryotes And Eukaryotes Prokaryote gene expression typically is regulated by an operon, the collection of controlling sites adjacent to protein-coding sequence. Eukaryotic genes also are regulated in units of proteincoding sequences and adjacent controlling sites, but operons are not known to occur. Eukaryotic gene regulation is more complex because eukaryotes possess a nucleus (transcription and translation are not coupled). Two “Categories” Of Eukaryotic Gene Regulation Short-term - genes are quickly turned on or off in response to the environment and demands of the cell. Long-term - genes for development and differentiation. Eukaryote Gene Expression Is Regulated At Six Levels Transcription RNA processing mRNA transport mRNA translation mRNA degradation Protein degradation Transcription Control Of Gene Regulation Controlled By Promoters Occur upstream of the transcription start site. Some determine where transcription begins (e.g., TATA), whereas others determine if transcription begins. Promoters are activated by highly specialized transcription factor (TF) proteins (specific TFs bind specific promoters). One or many promoters (each with specific TF proteins) may occur for any given gene. Promoters may be positively or negatively regulated. Transcription Control Of Gene Regulation Controlled By Enhancers Occur upstream or downstream of the transcription start site. Regulatory proteins bind specific enhancer sequences; binding is determined by the DNA sequence. Loops may form in DNA bound to TFs and make contact with enhancer elements. Interactions of regulatory proteins determine if transcription is activated or repressed (positively or negatively regulated). Chromosome Structure, Eukaryote Chromosomes, And Histones Prokaryotes lack histones and other structural proteins, so access to the DNA is straightforward. Eukaryotes possess histones, and histones repress transcription because they interfere with proteins that bind to DNA. Verified by DNase I sensitivity experiments: DNase I readily degrades transcriptionally active DNA. Histones shield non-transcribed DNA from DNase I, and DNA does not degrade as readily. Chromosome Structure, Eukaryote Chromosomes, And Histones If you experimentally add histones and promoter binding proteins; histones competitively bind to promoters and inhibit transcription. Solution: transcriptionally active genes possess looser chromosome structures than inactive genes. Histones are acetylated and phosphorylated, altering their ability to bind to DNA. Enhancer binding proteins competitively block histones if they are added experimentally with histones and promoterbinding TFs. RNA polymerase and TFs “step-around” the histones/nucloesome and transcription occurs. Genomic Imprinting (Silencing) Methylation of DNA inhibits transcription of some genes. Methylation usually occurs on cytosines or adenines. 5-methyl cytosine N-6 methyl adenine N-4 methyl cytosine CpG islands are sites of methylation in human DNA. CpG Island …ggaggagcgcgcggcggcggccagagaaaaa gccgcagcggcgcgcgcgcacccggacagccgg cggaggcggg... DNA Methylation And Transcription Control Small percentages of newly synthesized DNAs (~3% in mammals) are chemically modified by methylation. Methylation occurs most often in symmetrical CG sequences. Transcriptionally active genes possess significantly lower levels of methylated DNA than inactive genes. A gene for methylation is essential for development in mice (turning off a gene also can be important). Methylation results in a human disease called fragile X syndrome; FMR-1 gene is silenced by methylation. Hormone Regulation: Example Of Short-term Regulation Of Transcription Cells of higher eukaryotes are specialized and generally shielded from rapid changes in the external environment. Hormone signals are one mechanism for regulating transcription in response to demands of the environment. Hormones act as inducers produced by one cell and cause a physiological response in another cell. Hormone Regulation: Example Of Short-term Regulation Of Transcription Hormones act only on target cells with hormone specific receptors, and levels of hormones are maintained by feedback pathways. Hormones deliver signals in two different ways: Steroid hormones pass through the cell membrane and bind cytoplasmic receptors, which together bind directly to DNA and regulate gene expression. Polypeptide hormones bind at the cell surface and activate transmembrane enzymes to produce second messengers (such as cAMP) that activate gene transcription. Examples Of Mammalian Steroid Hormones Hormone Regulation Genes regulated by steroid hormones possess binding regions in the sequence called steroid hormone response elements (HREs). HREs often occur in multiple copies in enhancer sequence regions. When steroid is absent: Receptor is bound and “guarded” by chaperone proteins; transcription does not occur. When steroid is present: Steroid displaces the chaperone protein, binds the receptor, and binds the HRE sequence; transcription begins. Model Of Glucocorticoid Steroid Hormone Regulation RNA Processing Control RNA processing regulates mRNA production from precursor RNAs. Two independent regulatory mechanisms occur: Alternative polyadenylation = where the polyA tail is added Alternative splicing = which exons are spliced Alternative polyadenylation and splicing can occur together. Examples: Human calcitonin (CALC) gene in thyroid and neuronal cells Sex determination in Drosophila Alternative Polyadenylation And Splicing Of The Human CACL Gene In Thyroid And Neuronal Cells mRNA Transport Control Eukaryote mRNA transport is regulated. Some experiments show ~1/2 of primary transcripts never leave the nucleus and are degraded. Mature mRNAs exit through the nuclear pores. mRNA Transport Control Unfertilized eggs are an example, in which mRNAs (stored in the egg/no new mRNA synthesis) show increased translation after fertilization). Stored mRNAs are protected by proteins that inhibit translation. Poly(A) tails promote translation. Stored mRNAs usually have short poly(A) tails (15-90 As vs 100300 As). Specific mRNAs are marked for deadenylation (“tail-chopping”) prior to storage by AU-rich sequences in 3’-UTR. Activation occurs when an enzyme recognizes AU-rich element and adds ~150 As to create a full length poly(A) tail. mRNA Degradation Control All RNAs in the cytoplasm are subject to degradation. tRNAs and rRNAs usually are very stable; mRNAs vary considerably (minutes to months). Stability may change in response to regulatory signals and is thought to be a major regulatory control point. Various sequences and processes affect mRNA half-life: AU-rich elements Secondary structure Deadenylation enzymes remove As from poly(A) tail 5’ de-capping Internal cleavage of mRNA and fragment degradation Post-translational Control - Protein Degradation Proteins can be short-lived (e.g., steroid receptors) or long-lived (e.g., lens proteins in your eyes). Protein degradation in eukaryotes requires a protein cofactor called ubiquitin. Ubiquitin binds to proteins and identifies them for degradation by proteolytic enzymes. Amino acid at the N-terminus is correlated with protein stability and determines rate of ubiquitin binding. Arg, Lys, Phe, Leu, Trp Cys, Ala, Ser, Thr, Gly, Val, Pro, Met 1/2 life ≤3 minutes 1/2 life ≥ 20 hours Epigenetics Non-sequence specific, heritable traits Transcriptional gene silencing (TGS) Imprinting X-inactivation RNA-induced transcriptional silencing (RITS) Post-transcriptional gene silencing (PTGS) RNA-induced silencing complex (RISC) G quartets Post-translational protein-protein interactions RNAi First described in C. elegans Injecting antisense RNA into oocytes (ssRNA that is complementary to mRNA) Silences gene expression Also injected dsRNA into oocytes found it was 10X more potent in inhibiting expression Term RNAi RNAi Higher eukaryotes produce a class of small RNA’s that mediate silencing of some genes Small RNA’s interact with mRNA in the 3’UTR and this results in either mRNA degradation or translation inhibition Controls developmental timing in at least some organisms Used as a mechanism to protect against invading RNA viruses (plants) Controls the activity of transposons Formation of heterochromatin Mechanism of RNA Interference Step 1 Step 2 Nat Rev Genet. 2002 Oct;3(10):737-47. Review. Epigenetics The study of mechanisms by which genes bring about their phenotypic effects Epigenetic changes influence Phenotype without altering Genotype Changes in properties of a cell that are inherited but don’t represent a change in genetic information. A Histone Code? Modifications of histones (usually the amino terminal tails) convey epigenetic information Types of modifications: Acetylation and methylation of lysine. Methylation of arginine Phosphorylation of serine Ubiquitination of lysine. The histone code hypothesis posits that serial modifications provide a blueprint for reading chromatin -for transcription, replication, repair, and recombination. Remodeling Nucleosomes Changing the way nucleosomes bind to the DNA in chromosomes is important to allow access to the underlying DNA sequences during DNA replication, repair, recombination, and transcription. This occurs in three general ways: Modification of the lys and Arg residues on the histone tails decreases the grip of the nucleosome on DNA and causes the nucleosome to slide more easily. Variant histones are added to pre-existing nucleosomes ATP-dependent protein “remodeling”complexes cause nucleosomes to dissociate and/or slide along the DNA.