Download Transcription and the Central Dogma

Transcription and the Central Dogma
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• 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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About RNA
1) DNA is double stranded, but RNA
is single stranded.
However, RNA can base-pair with
itself to create double stranded
regions.
RNA
DNA
tRNA
genetics.gsk.com/graphics/ dna-big.gif http://www.fhi-berlin.mpg.de/th/JG/RNA.jpg
http://www.santafe.edu/images/rna.gif
About RNA-2
2) RNA contains ribose
instead of deoxyribose
3) RNA contains uracil
instead of thymine.
www.layevangelism.com/.../ deoxyribose.htm
http://www.rothamsted.bbsrc.ac.uk/notebook/courses/guide/images/uracil.gif
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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
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3 kinds of RNA
mRNA: a copy of the gene;
is translated to make protein.
tRNA: smallest RNA, does
actual decoding.
tRNA
rRNA: 3 sizes that,
along with proteins,
make up a ribosome.
rRNA
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
More kinds of RNA
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• 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
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mRNA, obviously
tRNA, rRNA
Telomerase guide RNA
Xist
antisense-RNA
Thus, we talk about RNA genes, sections of
DNA that code for RNAs
Using 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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Transcription: making mRNA
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• 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.
Transcription-2
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• 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.
Different genes on different strands
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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
Sense, antisense
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Compare the sense strand of the DNA to the mRNA.
Note that mRNA synthesis will be 5’ to 3’ and antiparallel.
DNA between genes has no sense.
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/SenseStrand.gif
More about RNA polymerase
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• 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)
Transcription needs a Promoter
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A promoter is nontranscribed DNA
Prokaryotes
Eukaryotes
http://opbs.okstate.edu/~petracek/2002%20Gene%20expression/img043.gif
The Process of Transcription
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• 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.
Consensus sequence
Numbers indicate the
percentage of different genes
in which that nucleotide
appears in that spot in the
promoter sequence.
http://www.uark.edu/campus-resources/mivey/m4233/promoter.gif
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The Process of Transcription-2
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• 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.
Termination of Transcription in Bacteria
In euks, termination
occurs with a
processing step.
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The hairpin loop
destabilizes the
interactions between
the DNA, mRNA, and
polymerase; U-A
basepairs are very
weak, and the
complex falls apart.
http://www.blc.arizona.edu/marty/411/Modules/Weaver/Chap6/Fig.0649ac.gif
About mRNA structure, etc.
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• 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.
Eukaryotic 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 premRNAs
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Initiation 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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Eukaryotic RNA polymerase
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• 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)
Processing 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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Processing 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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Introns 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
Removal 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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Splicing 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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Introns:
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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.
Why are there introns?
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• 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.
Relationship between protein domains and exons
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http://www.mrclmb.cam.ac.uk/genomes/cvogel/SupraDomains/Data_new/sd.figure.supra-domain.jpg