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Gene Expression: Nucleus DNA Pol II transcription Primary RNA transcript Multiple, Spatially and Temporally Distinct Steps Carried out by Distinct Cellular Machinery Nuclear processing Capping Splicing Polyadenylation Cytoplasm Mature mRNA Mature mRNA Export Degradation Protein Translation - Regulation Can Be at Several Different Levels - Dynamic Protein Association How can the cell distinguish between (1) intron-containing pre-mRNAs (2) spliced mRNAs (3) intronless mRNAs to ensure that (1) are retained in the nucleus while (2 and 3) are exported to the cytoplasm? Mechanisms of mRNA turnover (NMD, mRNAs contain a premature stop codon, an in-frame stop codon within a retained intron, or an extended 3’ UTR due to improper polyadenylation site usage) (mRNAs lack an in-frame termination codon-decay occurs in the cytoplasm) Nuclear retention of unspliced mRNAs •An intact 5’ splice site and branchpoint are required for nuclear retention of pre-mRNAs •Numerous splicing factors, including U1 snRNA and branchpoint binding protein (BBP/SF1), have been found to affect nuclear retention of pre-mRNAs •In yeast, perinuclearly located Mlp1 physically retains improperly spliced pre-mRNAs but does not affect the splicing process itself Thus, it appears that Mlp1 retains pre-mRNAs that assemble into a spliceosome but fail to proceed through splicing before reaching the nuclear pore complex green-Mlp1 detected with an antibody red-the nucleolar protein Nop1 detected with an antibody Galy, V. et al. Cell 116, 63-73 (2004) Coupling of Transcriptional and Post-Transcriptional Events mRNA Surveillance RNA-Mediated Gene Silencing Coupling of Transcriptional and Post-Transcriptional Events A) CTD of RNA Pol II - binding platform for mRNA processing components B) TREX - couples TRanscription and EXport C) Exon Junction Complex (EJC) - splicing mark and coupler mRNA Surveillance A) Quality Control Mechanism - ‘Process vs. Discard’ B) Nonsense Mediated Decay - Elimination of mRNAs with Premature Stop Codons (PTCs) RNA-Mediated Gene Silencing A) Post-Transcriptional Gene Silencing (PTGS or RNA Interference) - mRNA degradation - translation block B) Transcriptional Gene Silencing (TGS) - DNA methylation - Heterochromatin formation - DNA rearrangement/elimination Gene Expression: Nucleus Linear Assembly Line? DNA Pol II transcription Primary RNA transcript Nuclear processing Capping Splicing Polyadenylation Cytoplasm Mature mRNA Mature mRNA Export Degradation Protein Translation Gene Expression: Complex Network of Coupled Interactions Maniatis, T., and Reed , R. (2002). Nature 416, 499-506. Co-Transcriptional Recruitment of pre-mRNA Processing Factors TREX 5’ Nascent mRNA CTD 5’ Capping Enzymes o Splicing Machinery o 3’ Cleavage/PolyA Factors o Export Receptors o Jensen, T.H., Dowe r, K., Libri, D., and Ro sbash, M. (2003). Mol Cell 11, 1129-1138. Transcription Export T-REX Transcription Elongation Factors (THO Complex: Hpr1p, Tho2p, Mft1p, Thp2p) Export Factors (Yra, Sub2) Tex1 (unknown function) http://home.wxs.nl/~vrie0388/trex.JPG Nuclear mRNA Surveillance and Quality Control: Process or Discard Degradation by Nuclear Exosome (3’-5’ exonucleases) Transcriptional Coupling & mRNA Surveillance RNA Pol II Nascent mRNA NPC Exosome Jensen , T.H., and Rosb ash, M. (2003). Nat Struct Biol 10, 10-12. Ribosome Exon Junction Complex The Splicing Process Leaves its ‘Mark’ EJC • The EJC assembles following splicing. • EJC is deposited 20-24 nts upstream of the exon-exon junction. • EJC as a ‘Molecular Link’ between splicing and downstream events. EJC: A Molecular Link Between mRNA Splicing and Subsequent Events (Export, Localization, Decay, Translation, etc.) Tange, T.O., Nott, A., and Moo re, M.J. (2004). Curr Opin Cell Biol 16, 279-284. RNA-Mediated Gene Silencing RNA Interference (PTGS) Transcriptional Gene Silencing (TGS) Common Trigger: Mechanism of RNAi: Gene Silencing directed by ~22nt RNAs dsRNA DICER processing ~22nt siRNAs RISC target mRNA recognition (Argonaute) degradation RISC: RNA-Induced Silencing Complex (contains effector nuclease) MicroRNAs and SiRNAs: Small but Mighty ‘Riboregulators’! - Viruses - Transposons - Repeat Elements - Exogenous Endogenous Host genes MicroRNA Precursor Translational Repression mRNA Degradation Proposed Biologic Roles ‘Immune System’ of the Genome • Antiviral Defense • Suppress Transposon Activity • Gene Regulation (Silencing) (e.g. MicroRNAs, Heterochromatin) RNA-Mediated Gene Silencing RITS RNA-Induced Initiator of Transcriptional Gene Silencing (siRNAs, Ago1, Chp1 (chromodomain protein), Tas3) RITS RITS DNA methylation - Multiple dsRNA Inputs - Different Silencing Events Bartel, D.P. (2004). Cell 116, 281-297. Mechanism of RNAi-Mediated Heterochromatin Formation Trigger: Repetitive DNA RITS DNA 2. Recruitment Two Very Different Outcomes! 1. Histone 3 Methylation TRANSLATION Molecular Biology Familiarity with basic concepts is assumed, including: nature of the genetic code maintenance of genes through DNA replication transcription of information from DNA to mRNA translation of mRNA into protein. DNA mRNA protein Genetic code The genetic code is based on the sequence of bases along a nucleic acid. Each codon, a sequence of 3 bases in mRNA, codes for a particular amino acid, or for chain termination. Some amino acids are specified by 2 or more codons. Synonyms (multiple codons for the same amino acid) in most cases differ only in the 3rd base. Similar codons tend to code for similar amino acids. Thus effects of mutation are minimized. Genetic Code 1st base U UUU Phe U UUC Phe UUA Leu UUG Leu CUU Leu C CUC Leu CUA Leu CUG Leu AUU Ile A AUC Ile AUA Ile AUG Met* GUU Val G GUC Val GUA Val GUG Val *Met and initiation. 2nd base C UCU Ser UCC Ser UCA Ser UCG Ser CCU Pro CCC Pro CCA Pro CCG Pro ACU Thr ACC Thr ACA Thr ACG Thr GCU Ala GCC Ala GCA Ala GCG Ala A UAU Tyr UAC Tyr UAA Stop UAG Stop CAU His CAC His CAA Gln CAG Gln AAU Asn AAC Asn AAA Lys AAG Lys GAU Asp GAC Asp GAA Glu GAG Glu 3rd base G UGU Cys UGC Cys UGA Stop UGG Trp CGU Arg CGC Arg CGA Arg CGG Arg AGU Ser AGC Ser AGA Arg AGG Arg GGU Gly GGC Gly GGA Gly GGG Gly U C A G U C A G U C A G U C A G Prokaryotic genes Prokaryotes (intronless protein coding genes) Upstream (5’) promoter TAC Gene region Downstream (3’) DNA Transcription (gene is encoded on minus strand .. And the reverse complement is read into mRNA) ATG 5´ UTR CoDing Sequence (CDS) mRNA 3´ UTR ATG Translation: tRNA read off each codons, 3 bases at a time, starting at start codon until it reaches a STOP codon. protein Prokaryotic genes (operons) Prokaryotes (operon structure) upstream promoter downstream Gene 1 Gene 2 Gene 3 In prokaryotes, sometimes genes that are part of the same operational pathway are grouped together under a single promoter. They then produce a pre-mRNA which eventually produces 3 separates mRNA´s. Bacterial Gene Structure of signals - translation binding site (shine-dalgarno) 10 bp upstream of AUG (AGGAGG) - One or more Open Reading Frame •start-codon (unless sequence is partial) •until next in-frame stop codon on that strand .. Separated by intercistronic sequences. - Termination Eukaryotic Central Dogma In Eukaryotes ( cells where the DNA is sequestered in a separate nucleus) The DNA does not contain a duplicate of the coding gene, rather exons must be spliced. ( many eukaryotes genes contain no introns! .. Particularly true in ´lower´ organisms) mRNA – (messenger RNA) Contains the assembled copy of the gene. The mRNA acts as a messenger to carry the information stored in the DNA in the nucleus to the cytoplasm where the ribosomes can make it into protein. tRNA The genetic code is read during translation via adapter molecules, tRNAs, that have 3-base anticodons complementary to codons in mRNA. "Wobble" during reading of the mRNA allows some tRNAs to read multiple codons that differ only in the 3rd base. There are 61 codons specifying 20 amino acids. Minimally 31 tRNAs are required for translation, not counting the tRNA that codes for chain initiation. Mammalian cells produce more than 150 tRNAs. tRNA ( transfer RNA) is a small RNA that has a very specific secondary and tertiary structure such that it can bind an amino acid at one end, and mRNA at the other end. It acts as an adaptor to carry the amino acid elements of a protein to the appropriate place as coded for by the mRNA. T Secondary structure of tRNA Threedimensional Tertiary structure RNA structure: Most RNAs have secondary structure, consisting of stem & loop domains. A : U U : A A : U stem C : G C UG C U : G U C U loop Double helical stems arise from base pairing between complementary stretches of bases within the same strand. Loops occur where lack of complementarity, or the presence of modified bases, prevents base pairing. anticodon loop The “cloverleaf” model of tRNA secondary structure emphasizes the 2 major types of secondary structure, stem and loop domains. tRNA acceptor stem tRNAs typically include many modified bases, particularly in the loop domains. Tertiary structure depends on interactions of bases at more distant sites. Many of these interactions involve non-standard base pairing and/or interactions involving three or more bases. tRNAs usually fold into an L-shaped tertiary structure. anticodon loop tRNA Extending out from the "acceptor stem", the 3' end of every tRNA has the sequence CCA. acceptor stem The appropriate amino acid is attached to the ribose of the terminal A (in red) at the 3' end. The anticodon loop is at the opposite end of the L shape. anticodon Phe tRNA acceptor stem Tertiary base pairs #46 (m7G) #22 G Tertiary base pairs in tRNAPhe #13 C #46 (m7G) #22 G #13 C Tertiary base pairs in tRNAPhe Non-standard H bond interactions, some linking 3 bases, help stabilize the L-shaped tertiary structure of tRNA. This example is from NDB file 1TN2. H atoms are not shown. O R H C C O O O P Amino acid P C O O P O O CH2 O H ATP O R O O NH3+ H C O Adenine O H H OH H OH O O NH2 Aminoacyl-AMP P O CH2 O H Adenine O H H OH H OH PPi Aminoacyl-tRNA Synthetases catalyze linkage of the appropriate amino acid to each tRNA. The reaction occurs in two steps. In step 1, an O atom of the amino acid a-carboxyl attacks the P atom of the initial phosphate of ATP. O R H C C O O P O CH2 O NH2 H Aminoacyl-AMP In step 2, the 2' or 3' OH of the terminal adenosine of tRNA attacks the amino acid carbonyl C atom. O H H OH H OH tRNA AMP tRNA Adenine O O P O CH2 O Adenine O H H H 3’ 2’ H OH O C O HC R NH3+ (terminal 3’nucleotide of appropriate tRNA) Aminoacyl-tRNA Aminoacyl-tRNA Synthetase Summary of the 2-step reaction: 1. amino acid + ATP aminoacyl-AMP + PPi 2. aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP The 2-step reaction is spontaneous overall, because the concentration of PPi is kept low by its hydrolysis, catalyzed by Pyrophosphatase. There is a different Aminoacyl-tRNA Synthetase (aaRS) for each amino acid. Each aaRS recognizes its particular amino acid and the tRNAs coding for that amino acid. Accurate translation of the genetic code depends on attachment of each amino acid to an appropriate tRNA. Domains of tRNA recognized by an aaRS are called identity elements. Most identity elements are in the anticodon loop acceptor stem & anticodon loop. Aminoacyl-tRNA Synthetases arose early in evolution. The earliest aaRSs probably recognized tRNAs acceptor only by their acceptor stems. stem tRNA tRNA O O P O (terminal 3’nucleotide of appropriate tRNA) O O H H There are 2 families of Aminoacyl-tRNA Synthetases: Class I & Class II. Adenine CH2 O H 3’ 2’ H OH C O HC R NH3+ Aminoacyl-tRNA Two different ancestral proteins evolved into the 2 classes of aaRS enzymes, which differ in the architecture of their active site domains. They bind to opposite sides of the tRNA acceptor stem, resulting in aminoacylation of a different OH of the tRNA (2' or 3'). Class I aaRSs: Identity elements usually include residues of the anticodon loop & acceptor stem. Class I aaRSs aminoacylate the 2'-OH of adenosine at their 3' end. Class II aaRSs: Identity elements for some Class II enzymes do not include the anticodon domain. Class II aaRSs tend to aminoacylate the 3'-OH of adenosine at their 3' end. Proofreading/quality control: Some Aminoacyl-tRNA Synthetases are known to have separate catalytic sites that release by hydrolysis inappropriate amino acids that are misacylated or mistransferred to tRNA. E.g., the aa-tRNA Synthetase for isoleucine (IleRS) a small percentage of the time activates the closely related amino acid valine to valine-AMP. After valine is transferred to tRNAIle, to form Val-tRNAIle, it is removed by hydrolysis at a separate active site of IleRS that accommodates Val but not the larger Ile. In some bacteria, editing of some misacylated tRNAs is carried out by separate proteins that may be evolutionary precursors to editing domains of aa-tRNA Synthetases. Some amino acids are modified after being linked to tRNA. E.g., in prokaryotes & in mitochondria the initiator tRNAfMet is first charged with methionine. Methionyl-tRNA formyltransferase then catalyzes formylation of the methionine moiety, using THF as formyl donor, to yield fMet-tRNAfMet. In some prokaryotes, a non-discriminating aaRS loads aspartate onto tRNAAsn. The aspartate moiety is then converted by an amidotransferase to asparagine, yielding Asn-tRNAAsn. Glu-tRNAGln is similarly formed and converted to GlntRNAGln in such organisms. RIBOSOMES Ribosome Composition (S = sedimentation coefficient) Ribosome Source E. coli Whole Ribosome 70S Small Subunit 30S 16S RNA 21 proteins Rat cytoplasm 80S 40S 18S RNA 33 proteins Large Subunit 50S 23S & 5S RNAs 31 proteins 60S 28S, 5.8S, &5S RNAs 49 proteins Eukaryotic cytoplasmic ribosomes are larger and more complex than prokaryotic ribosomes. Mitochondrial and chloroplast ribosomes differ from both examples shown. 5S rRNA “crown” view displayed as ribbons & sticks. PDB 1FFK Structures of large & small subunits of bacterial & eukaryotic ribosomes have been determined, by X-ray crystallography & by cryo-EM with image reconstruction. Consistent with predicted base pairing, X-ray crystal structures indicate that ribosomal RNAs (rRNAs) have extensive secondary structure. Structure of the E. coli Ribosome large subunit tRNA EF-G small subunit mRNA location The cutaway view at right shows positions of tRNA (P, E sites) & mRNA (as orange beads). EF-G will be discussed later. This figure was provided by Joachim Frank, whose lab at the Wadsworth Center carried out the cryo-EM and 3D image reconstruction on which the images are based. Small Ribosomal Subunit In the translation complex, mRNA threads through a tunnel in the small ribosomal subunit. tRNA binding sites are in a cleft in the small subunit. The 3' end of the 16S rRNA of the bacterial small subunit is involved in mRNA binding. The small ribosomal subunit is relatively flexible, assuming different conformations. E.g., the 30S subunit of a bacterial ribosome was found to undergo specific conformational changes when interacting with a translation initiation factor. Small ribosomal subunit of a thermophilic bacterium: rRNA in monochrome; proteins in varied colors. 30S ribosomal subunit spacefill display PDB 1FJF ribbons The overall shape of the 30S ribosomal subunit is largely determined by the rRNA. The rRNA mainly consists of double helices (stems) connected by single-stranded loops. The proteins generally have globular domains, as well as long extensions that interact with rRNA and may stabilize interactions between RNA helices. Large ribosome subunit: The interior of the large subunit is mostly RNA. Proteins are distributed mainly on the surface. PDB 1FFK Large Ribosome Subunit Some proteins have long tails that extend into the interior of the complex. These tails, which are highly basic, interact with the negatively charged RNA. "Crown" view with RNAs blue, in spacefill; proteins red, as backbone. The active site domain for peptide bond formation is essentially devoid of protein. PDB 1FFK Large Ribosome Subunit Peptidyl transferase is attributed to 23S rRNA, making this RNA a "ribozyme." A universally conserved adenosine base serves as a general acid base during peptide bond formation. "Crown" view with RNAs blue, in spacefill; proteins red, as backbone. Protein synthesis takes PDB 1FFK place in a cavity within the ribosome. Nascent polypeptides emerge through a tunnel in the large subunit. Some nascent proteins then pass through a channel into the ER lumen, or across the cytoplasmic membrane Large ribosome subunit. Backbone display with RNAs blue. View and out of the cell in from bottom at tunnel exit. prokaryotes. small subunit Sec61 channel large subunit path of nascent protein The cutaway view at right shows that the tunnel in the yeast large ribosome subunit, through which nascent polypeptides emerge from the ribosome, lines up with the lumen of the ER Sec61 channel.