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1. 2. 3. 4. The role of RNA in protein synthesis RNA polymerase Control of transcription in eukaryotes Posttranscriptional processing
Historically
DNA found in cell nucleus, but RNA found in cytosol (1930), microscopy and cell fractionation Concentration of cytosolic RNA-Protein particles correlate with protein synthesis - is site of protein synthesis - was later identified as Ribosome In eukaryotes, DNA is never in association with protein synthesis (Ribosome) Incorporation of radiolabelled amino acids occurs in association with RNA-Protein particles Structure of DNA revealed possible copy mechanism
Central Dogma
DNA RNA Protein
Enzyme induction
E. coli can synthesize 4300 polypeptides
But enormous variation in abundance of specific polypeptides: Ribosomal protein: 10000 copies/cell Regulatory protein: <10 copies/cell Housekeeping enzymes, constitutive Adaptive, inducible enzymes
Structural genes
Control site
Regulatory gene
I, inducer
P, promoter O, operator
Bacterial conjugation
Transfer of the bacterial chromosome from an Hfr cell to an F cell and its subsequent recombination with the F chromosome
F factor can spontaneously integrate into the genome -> Hfr strain (high frequency of recombination) -> transmission of genomic information upon mating: in fixed order time dependent (90min) Merozygote, partially diploid Recombination and integration
Induction after 1h, cessation upon 2h -> Z+ in I- cells leads to constitutive induction, cessation upon transfer of I gene -> I gene codes for diffusible repressor of Z, lac repressor F- is resistant to T6 and streptomycin
Messenger RNA
Second type of constitutive mutation Oc, operator constitutive, maps between I and Z genes In merozygote F Oc Z- / F O+ Z+, -gal inducible But in Oc Z+ / F O+ Z- constitutive synth of -gal O Can control Z only when on same chromosome !!! -> cis acting control I is trans acting factor Proteins are synthesized in two stages: 1. DNA is transcribed in mRNA 2. mRNA is translated into protein This model explains behavior of lac system
Messenger RNA
In the absence of inducer, I binds to O and represses synthesis of structural genes Z, Y, A On binding inducer, repressor dissociates from O, permitting transcription and subsequent translation Operator-Repressor-Inducer system represents a molecular switch Oc is constitutive because repressor cannot bind, Cis-acting element Coordination of all 3 protein by single polycistronic mRNA transcript, cistron
The distribution, in a CsCl density gradient, of 32P-labeled RNA that had been synthesized by E. coli after T4 phage infection
The hybridization of 32P-labeled RNA produced by T2-infected E. coli with 3H-labeled T2 DNA
2. RNA Polymerase
RNA polymerase is responsible for the DNA-directed synthesis of RNA (1960), dNTP and DNA-dep. E. coli RNAP hplpenzyme, 459kD, ' subunit composition, sigma 70 unit dissociates from core once RNA synthesis has been initiated RNAP functions: 1. Template binding 2. RNA chain initiation 3. Chain elongation 4. Chain termination
Electron micrograph of E. coli RNA polymerase (RNAP) holoenzyme attached to various promoter sites on bacteriophage T7 DNA
Template binding
o RNA synthesis is initiated only at specific sites on the DNA template o RNAP binds to its initiation sites at sequence elements called promoter, these are recognized by sigma factor (K 10-14M) o Promoter, ca. 40bp element, located 5 of structural gene, first base in RNA is +1, initiation site o If RNAP bound to promoter -20 to +20 are DNaseI protected o Consensus promoter sequence, hexamer centered at 10 = Pribnow Box, TATAAT, plus additional element at 35 sequence in between not important, but distance o +1 is either A or G
Rate of transcription varies 1000-fold, correlates with strength of promoter to bind RNAP
Chain initiation
+1 is purine, A > G Initiation reaction: pppA + pppN -> pppApN + Ppi Unlike DNA replication, RNA initiation does not need a primer Crab claw shape of Taq RNAP, pincers formed by and with a cavern between the two pincers Prokaryotic initiation is inhibited by rifamycin B, Steroptomyces mediterranei, rifampicin commercial grampositive antibiotic (also tubeculosis), does not block promoter bdg or elongation, but only initiation
Chain elongation
5 -> 3 or 3 -> 5 growth ? Label with [32P]GTP, chase with cold GTP If 5->3, RNA is permanently labelled If 3->5, RNA is unlabelled
Chain Termination
EM specific sites of termination, two common features in E. coli: 1. Series of 4-10 consecutive A-Ts, As on template 2. G + C-rich palindromic region upstream of A-Ts
The stability of terminator G+C hairpin + weak base pairing Between RNA polyU and DNA template ensure termination
X-Ray structure of yeast RNAP II that lacks its Rpb4 and Rpb7 subunits
Resembles Taq RNAP crab claw like shape
Enhancers are transcriptional activators that can have variable positions and orientations
Promoter element that act in both orientation and distance independent = enhancers Can act from several kb, in euk. Viruses or structural genes Required for full activity of promoter Recognized by specific transcription factors -> DNA loop Stimulate entry of RNAP II on promoter Mediate much of selective gene expression
RNAP III promoters canbe located downstream from their transcription start site
RNAP III promoters can be within the genes transcribed region !! 5S rRNA
Promoters
Genes that are transcribed at high rates have efficient promoters Lac I is transcribed at 10 copies/cell
lac repressor prevents RNA polymerase from forming a productive initiation complex
RNA polymerase binds +20 to -20 Operator occupies +28 to -7 -> lac operator and promoter overlap Binding of repressor obstructs RNAP binding
The kinetics of lac operon mRNA synthesis following its induction with IPTG, and of its degradation after glucose addition
cAMP is second messenger in animal cells In E. coli, cAMP greatly diminished in presence of glucose Addition of cAMP to culture overcomes catabolite repression
cAMP binding protein = catabolite gene activator protein, CAP = cAMP receptor protein, CRP Homodimer of 210 Aa, undergoes large conformational change upon cAMP bdg. CAP-cAMP binds lac operon and stimulates transcription -> positive regulator (unlike lac I, negative regulator) Binds DNA and bends it 90, contacts CTD of RNAP
HTH-DNA interaction
Model of the 93-bp DNA loop formed when lac repressor binds to O1 and O3
DNA loop is further stabilized by cAMP-CAP binding Principal: Modular build up and break down of very high affinity complexes
4. Posttranscriptional Processing
Primary transcripts - of eukaryotes - are not yet functional but undergo post-transcriptional modifications: 1. Exo- and endonucleolytic removal of nt 2. Appending nt at 3 and 5 ends 3. Modification of specific nucleosides
Primary transcripts are heterogenous in size and much larger than mature mRNAs !! ->heterogenous nuclear RNA (hnRNA) mature to mRNAs by excision of internal sequences (pre-mRNA) Intervening sequence = intron (~1500nt, average 8/gene) Expressed sequence = exon (~300nt) Largest gene, titin 29926 Aa, 234 introns, 17kb exon
The sequence of transesterification reactions that splice together the exons of eukaryotic pre-mRNAs
Types of Introns
Group II introns
Mitochondria of fungi and plants Self splicing, lariat intermediate but no external nucleotide Spliceosome is an RNA-protein complex that mediates splicing of normal pre-mRNA, evolved from group primordial self-splicing RNA, Protein thought to be important for fine-tuning of ribozyme structure, Similar, RNA of ribosome has catalytic activity => RNA world hypothesis
Figure 31-55 The X-ray structure of the catalytic pocket in the hammerhead ribozymes kinetically trapped intermediate.
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A schematic diagram of six rearrangements that the spliceosome undergoes in mediating the first transesterification reaction in pre-mRNA splicing
1. 2. 3. 4. 5. 6.
Exchange of U1 for U6 in base pairing to 5 splice site Exchange of BBP for U2 in binding to branche site Intramolecular rearrangement in U2 Disruption of pairing between U4 and U6 Disruption of a second stem between U4 and U6 Disruption of a stem-loop in U2
Structure of the RNA binding portion of human branch point-binding protein (BBP) in complex with its target RNA
Spliceosomal structures
o All 4 snRNPs involved in pre-mRNA splicing contain the same snRNP core protein, which consist of 7 Sm proteins (react with autoantibodies from patients with systemic erythematosis), named B/B, D1, D2, D3, E. F, and G protein o Each of these Sm proteins contain two conserved segments, Sm1 and Sm2 separated by a variable linker o The seven Sm proteins bind to conserved RNA sequence, the SM RNA motif, occurs in U1-, U2-, U4, and U5-snRNA o Form heptameric ring, central hole positive charged allows passage of ss RNA but not ds RNA o U1-snRNP consist of 10 proteins, 7 Sm proteins and 3 U1 specific factors
Many eukaryotic proteins consist of modules that also occur in other proteins
o Example, LDL-receptor 839 Aa, 45kb gene, 18 exons, most encode specific functional domains, 13 of these segments/domains have homology with domains found in other proteins => modular construction of the LDL receptor => modular construction is found in many other proteins that are composed of re-utilized domains (i.e. signal transduction, SH2, SH3 domains etc.)
Alternative splicing greatly increases the number of proteins encoded by eukaryotic genome
o The expression of numerous cellular genes is modulated by the selection of alternative splice sites o Example rat -tropomyosin gene encodes 7 tissue specific variants of the muscle protein
Alternative splicing
o Occurs in all metazoans o Human genome only 30000 genes but estimated 50000 140000 structural genes o Entire functional domains or single amino acids can be altered in proteins through alternative splicing - soluble or membrane bound - can be phosphorylated by a specific kinase or not - subcellular localization - whether enzyme binds a specific allosteric effector - affinity of receptor - ligand interaction o Selection of alternative splice sites is developmental and tissue specific (regulation in space and time)
no TRA protein
Male-specific DSX protein -> represses female-specific genes Female-specific DSX protein -> represses male-specific genes (activation fo splice site)
Trans-splicing
o Trans-splicing, joining of two separate RNA molecules - Observed in Trypanosomas, all mRNAs have same 35nt leader, but this leader is not present in the corresponding genes - Splice leader (SL) RNA, transcribed from an independent gene - Trans-splicing reaction resembles spliceosome-mediated cis-splicing - But Y-shaped rather than lariat intermediate
A schematic diagram indicating how gRNAs direct the editing of trypanosomal pre-edited mRNAs
RNA editing occurs on ~20S RNP, editosome gRNA is used as template to correct the mRNA Requires: 1. Endonuclease 2. Terminal uridylyltransferase 3. RNA ligase
RNA interference
o Noncoding RNAs can have important roles in controlling gene expression o Anti-sense RNA can block translation of a specific message Yet injection of sense RNA into C. elegans also blocks protein production o Added RNA interferes with gene expression = RNA interference, RNAi o 1998 Andrew Fire and Craig Mello, Nobelprice 2006 ds RNA is substantially more efficient in causing RNAi induced by only a few molecules -> catalytic rather than stoichometric
Figure 31-74a The structure of the RNA of B. subtilis RNase P. (a) Predicted secondary structure with specificity domain drawn in various colors and catalytic domain is black.
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Figure 31-74b The structure of the RNA of B. subtilis RNase P. (b) The X-ray structure of the specificity domain in which its various segments are colored as in Part a.
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