Transcription and the Central Dogma
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1Transcription and the Central Dogma
• 1961: DNA is the molecule which stores genetic information, but– DNA is in nucleus, ribosomes (where protein
synthesis takes place) are in the cytoplasm.– RNA, a different nucleic acid, is synthesized in the
nucleus, and is similar to DNA.– RNA migrates to cytoplasm (where ribosomes are)– Amount of RNA generally proportional to amount of
proteins in the cell.
• All this suggests role of RNA as messenger.
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2About RNA
genetics.gsk.com/graphics/ dna-big.gif http://www.fhi-berlin.mpg.de/th/JG/RNA.jpg
http://www.santafe.edu/images/rna.gif
1) DNA is double stranded, but RNA is single stranded.
However, RNA can base-pair with itself to create double stranded regions.
DNA
RNA
tRNA
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3About RNA-2
www.layevangelism.com/.../ deoxyribose.htm http://www.rothamsted.bbsrc.ac.uk/notebook/courses/guide/images/uracil.gif
2) RNA contains ribose instead of deoxyribose
3) RNA contains uracil instead of thymine.
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RNA is similar to DNA
• Has sugar-phosphate backbone w/ nitrogenous bases
• Has a 5’ to 3’ directionality• When base pairing with
itself or other nucleic acids, is antiparallel
4
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53 kinds of RNA
http://www.cu.lu/labext/rcms/cppe/traducti/tjpeg/trna.jpeg;
Tobin and Duschek, Asking About Life; http://www.tokyo-ed.ac.jp/genet/mutation/nort.gif
mRNA: a copy of the gene; is translated to make protein.
tRNA: smallest RNA, does actual decoding.
rRNA: 3 sizes that, along with proteins, make up a ribosome.
tRNArRNA
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6More kinds of RNA
• snRNAs (small nuclear)
– Small RNAs (100-200 bases) that attach to proteins to form snRNPs (small nuclear ribonucleoproteins)• snRNPs are components of the spliceosome• Splicesome removes introns from pre-mRNA
• snoRNAs– Small nucleolar RNAs, process rRNA– Modify other RNAs; guide RNA of telomerase
• Other RNAs, e.g. Xist
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All RNA comes from the DNA
• mRNA, obviously
• tRNA, rRNA
• Telomerase guide RNA
• Xist
• antisense-RNA
• Thus, we talk about RNA genes, sections of DNA that code for RNAs
7
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8Using genetic information:the “central dogma”
Proteins are needed for cell structures, signaling, and as enzymes to carry out metabolism and regulation of processes.The information in encoded in the DNA; to use it requires transcription followed by translation.
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9Transcription: making mRNA
• RNA a polymer assembled from monomers– Ribonucleoside triphosphates: ATP, UTP, GTP,CTP
• RNA polymerase– Multi-component enzyme– Needs a template, but NOT a primer– In bacteria, a component (sigma) recognizes the
promoter as the place on DNA to start synthesis– Synthesis proceeds 5’ to 3’, just as in DNA
• mRNA is complementary and antiparallel to the DNA strand being copied.
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10Transcription-2
• The order of nucleotides in the RNA reflects the order in the DNA
• If RNA is complementary to one DNA strand, then it is identical (except for T change to U) to the other DNA strand.
Either DNA strand may contain the gene! Transcription just runs the other direction.
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Different genes on different strands11
A blow up of a section of DNA in the plant Arabidopsis.Genes above the line are transcribed left to right (on one strand), those below the line are transcribed right to left (on the other strand).http://www.sciencemag.org/feature/data/1051477s1_large.jpeg
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12Sense, antisense
Compare the sense strand of the DNA to the mRNA.
Note that mRNA synthesis will be 5’ to 3’ and antiparallel.
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/SenseStrand.gif
DNA between genes has no sense.
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13More about RNA polymerase
• In bacteria, components are ββ’α2φσ
• RNA polymerase is processive; once enzyme attaches to DNA, it can copy >10,000 nucleotides without falling off.
• In eukaryotes, there are 3 RNA polymerases:– One for rRNA– One for tRNAs and some rRNA– One for all mRNAs and some small RNAs (involved
in RNA processing)
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14Transcription needs a Promoter
http://opbs.okstate.edu/~petracek/2002%20Gene%20expression/img043.gif
A promoter is non-transcribed DNA
Prokaryotes
Eukaryotes
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15The Process of Transcription
• Promoter recognition: 2 consensus sequences– The -10 region: TATAAT (10 bases upstream from
where transcription actually starts.– The -35 region, farther upstream, also important.– “Consensus” sequence meaning the DNA sequence
from many genes averages out to this.– The closer these 2 regions actually are to the
consensus sequences, the “stronger” the promoter, meaning the more likely RNA polymerase binding and transcription will occur.
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16Consensus sequence
http://www.uark.edu/campus-resources/mivey/m4233/promoter.gif
Numbers indicate the percentage of different genes in which that nucleotide appears in that spot in the promoter sequence.
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17The Process of Transcription-2
• After binding to the promoter, polymerase “melts” DNA, lines up first base at the +1 site = Initiation.
• RNA synthesis continues (Elongation), only the template strand being transcribed.
• Termination: must be a stop sign, right?– In bacteria, hairpin loop followed by run of U’s in
the RNA. Of course, the DNA must code for complementary bases and a run of A’s. See next.
– Termination factor “rho”. Accessory protein.
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18Termination of Transcription in Bacteria
http://www.blc.arizona.edu/marty/411/Modules/Weaver/Chap6/Fig.0649ac.gif
The hairpin loop destabilizes the interactions between the DNA, mRNA, and polymerase; U-A basepairs are very weak, and the complex falls apart.
In euks, termination occurs with a processing step.
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19About mRNA structure, etc.
• Start site of transcription is NOT equal to start site of Translation– First codon read, AUG, is downstream from the first
ribonucleotides. +1 is transcription start, not translation start.
– AUG marks the beginning of an Open Reading Frame (ORF).
• Lifetime of a eukaryotic mRNA is variable• For prokaryotes, mRNA is short lived, fits in
with need of microbes to respond quickly to changes in environment.
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20Eukaryotic transcription
• Occurs in nucleus, then mRNA goes to cytoplasm
• Promoters but also enhancers– Enhancers also segments of DNA
• Eukaryotic RNA requires processing– Pre-mRNAs found in nucleus as hnRNA
• Heterogeneous nuclear RNA• hnRNPs: heterogeneous nuclear
ribonucleoproteins which bind to, process pre-mRNAs
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21Initiation of transcription (euks)
• Goldberg-Hogness (TATA) box at -35• CAAT box at -80 (non specific, but important)
– GGCCAATCT consensus sequence• Enhancers
– Elements of DNA that promote transcription– Can be upstream, downstream, even in gene
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22Eukaryotic RNA polymerase
• A 10 subunit machine
• Makes several attempts, abortive transcription
• Key: successful synthesis of short DNA-RNA hybrid, stabilizes association of Pol with DNA– Then, processive to a high degree, transcribes until
knocked off by termination signal.– Termination not well understood, happens along
with polyadenylation (adding poly-A tail, ahead 2)
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23Processing of mRNA
• Cap– 7-methyl guanosine is added as a cap, 5’ to 5’– Cap aids in binding of mRNA to the ribosome– Shine-Delgarno seq. does same in prokaryotes
http://www.blc.arizona.edu/Marty/429/Lectures/Figures/CAP.GIF
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24Processing of mRNA-2
• Poly-A tail– In several steps, end
of mRNA is cleaved off, and several rounds of AAAAAAA are added
– Poly-A tail improves stability of mRNA, resists degradation by nucleases in cell.
departments.oxy.edu/.../ processing_of_hnrnas.htm
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25Introns and Exons
•Introns are intervening sequences that do not contain information for making the protein.
Exons are the coding sequences left behind.
http://www.emc.maricopa.edu/faculty/farabee/BIOBK/exintrons.gif
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26Removal of Introns
• Three types of intron-removal mechanisms– Spliceosomes:
• hnRNA or pre-mRNA (to be processed)• snRNPs: small nuclear ribonucleoproteins• snRNAs: involved structurally and catalytically
– In some systems, intron codes for protein that splices out the intron!
– In Tetrahymena, intron is self-splicing: ribozyme
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27Splicing of Introns
• In most systems, snRNPs cut mRNA, introns come out, and snRNPs help splice exons back together.
• Particular sequences in the mRNA mark the beginning, end of exons and introns so snRNPs can do their job.
http://www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/splice1.GIF
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28Introns:
How were they discovered? RNA-DNA hybrids weren’t colinear- loops of DNA extend out where there is no RNA to base pair with it. RNA = red.
Mutations in introns: don’t have much affect unless:
•Mutation is near a splice site
•Mutation is in a regulatory region (which could be in an intron)
•There is a separate gene within the intron.
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29Why are there introns?
• Very ancient? – Located in the same positions in genes common to
plants and animals. Maybe bacteria had them once and lost them.
– Self-splicing RNAs may be related to RNA as the first nucleic acid, a popular idea in evolution.
• Exon-shuffling: a model for gene evolution– Some proteins fold into connected, functional
sections called domains; these correspond to exons– Perhaps exons were copied, shuffled to create new
genes. Several human genes share exons.
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30
http://www.mrc-lmb.cam.ac.uk/genomes/cvogel/SupraDomains/Data_new/sd.figure.supra-domain.jpg
Relationship between protein domains and exons