unit 1 lecture-genes
TRANSCRIPT
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Transcription and Translation
The Relationship
Between Genes and
Proteins
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Table of Contents
History: linking genes and proteins
Getting from gene to protein: transcription Evidence for mRNA
Overview of transcription
RNA polymerase
Stages of Transcription Promoter recognition
Chain initiation
Chain elongation
Chain termination
mRNA Synthesis/Processing References
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Table of Contents (continued)
Getting from gene to protein: genetic code
Getting from gene to protein: translation Translation Initiation
Translation Elongation
Translation Termination
References
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History: linking genes and proteins
1900s Archibald Garrod
Inborn errors of metabolism: inherited human metabolic diseases(more information)
Genes are the inherited factors
Enzymes are the biological molecules that drive
metabolic reactions
Enzymes are proteins
Question:
How do the inherited factors, the genes, control the structure and
activity of enzymes (proteins)?
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History: linking genes and proteins
Beadle and Tatum (1941) PNAS USA 27, 499506.
Hypothesis: If genes control structure and activity of metabolic enzymes, then
mutations in genes should disrupt production of required nutrients,
and that disruption should be heritable.
Method:
Isolated ~2,000 strains from single irradiate spores (Neurospora)
that grew on rich but not minimal medium. Examples: defects in B1,
B6 synthesis.
Conclusion:
Genes govern the ability to synthesize amino acids, purines andvitamins.
http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16588492http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16588492http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16588492http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16588492http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16588492http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16588492 -
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History: linking genes and proteins
1950s: sickle-cell anemia
Glu to Val change in hemoglobin Sequence of nucleotides in gene determines sequence of amino
acids in protein
Single amino acid change can alter the function of the protein
Tryptophan synthase gene in E. coli
Mutations resulted in single amino acid change
Order of mutations in gene same as order of affected amino acids
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From gene to protein: transcription
Gene sequence (DNA) recopied or transcribed to RNA
sequence Product of transcription is a messenger molecule that
delivers the genetic instructions to the protein synthesis
machinery: messenger RNA (mRNA)
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Transcription: evidence for mRNA
Brenner, S., Jacob, F. and Meselson, M. (1961) Nature
190, 57681. Question: How do genes work?
Does each one encode a different type of ribosome which in turn
synthesizes a different protein, OR
Are all ribosomes alike, receiving the genetic information to create
each different protein via some kind of messenger molecule?
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Transcription: evidence for mRNA
E. colicells switch from making bacterial proteins to
phage proteins when infected with bacteriophage T4. Grow bacteria on medium containing heavy nitrogen
(15N) and carbon (13C).
Infect with phage T4.
Immediately transfer to light medium containingradioactive uracil.
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Transcription: evidence for mRNA
If genes encode different ribosomes, the newly
synthesized phage ribosomes will be light. If genes direct new RNA synthesis, the RNA will contain
radiolabeled uracil.
Results:
Ribosomes from phage-infected cells were heavy, banding at thesame density on a CsCl gradient as the original ribosomes.
Newly synthesized RNA was associated with the heavy ribosomes.
New RNA hybridized with viral ssDNA, not bacterial ssDNA.
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Transcription: evidence for mRNA
Conclusion
Expression of phage DNA results in new phage-specific RNAmolecules (mRNA)
These mRNA molecules are temporarily associated with ribosomes
Ribosomes do not themselves contain the genetic directions for
assembling individual proteins
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Transcription: overview
Transcription requires:
ribonucleoside 5 triphosphates:ATP, GTP, CTP and UTP
bases are adenine, guanine, cytosine and uracil
sugar is ribose (not deoxyribose)
DNA-dependent RNA polymerase Template (sense) DNA strand
Animation of transcription
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Transcription: overview
Features of transcription:
RNA polymerasecatalyzes sugar-phosphate bondbetween 3-OH of ribose and the 5-PO4.
Order of bases in DNA template strand determines order
of bases in transcript.
Nucleotides are added to the 3-OH of the growing chain. RNA synthesis does not require a primer.
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Transcription: overview
In prokaryotes transcription and translation are coupled.
Proteins are synthesized directly from the primarytranscript as it is made.
In eukaryotes transcription and translation are separated.
Transcription occurs in the nucleus, and translation occurs
in the cytoplasm on ribosomes. Figurecomparing eukaryotic and prokaryotic transcription
and translation.
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Transcription: RNA Polymerase
DNA-dependent
DNA template, ribonucleoside 5 triphosphates, and Mg2+
Synthesizes RNA in 5 to 3 direction
E. coliRNA polymerase consists of 5 subunits
Eukaryotes have three RNA polymerases
RNA polymerase II is responsible for transcription of protein-codinggenes and some snRNA molecules
RNA polymerase II has 12 subunits
Requires accessory proteins (transcription factors)
Does not require a primer
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Stages of Transcription
Promoter Recognition
Chain Initiation Chain Elongation
Chain Termination
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Transcription: promoter recognition
Transcription factors bind to promoter sequences and
recruit RNA polymerase. DNA is bound first in a closed complex. Then, RNA
polymerase denatures a 1215 bp segment of the DNA
(open complex).
The site where the first base is incorporated into thetranscription is numbered +1 and is called the
transcription start site.
Transcription factors that are required at every promoter
site for RNA polymerase interaction are called basaltranscription factors.
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.View..ShowSection&rid=dbio.figgrp.752http://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.View..ShowSection&rid=dbio.figgrp.752http://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.View..ShowSection&rid=dbio.figgrp.752http://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.View..ShowSection&rid=dbio.figgrp.752 -
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Promoter recognition: promoter sequences
Promoter sequences vary considerably.
RNA polymerase binds to different promoters withdifferent strengths; binding strength relates to the level of
gene expression
There are some common consensus sequencesfor
promoters: Example: E. coli35 sequence (found 35 bases 5 to the start of
transcription)
Example: E. coliTATA box (found 10 bases 5 to the start of
transcription)
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Promoter recognition: enhancers
Eukaryotic genes may also have enhancers.
Enhancers can be locatedat great distances from thegene they regulate, either 5 or 3 of the transcription
start, in introns or even on the noncoding strand.
One of the most common ways to identify promoters and
enhancers is to use a reporter gene.
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Promoter recognition: other players
Many proteins can regulate gene expression by
modulating the strength of interaction between thepromoter and RNA polymerase.
Some proteins can activate transcription (upregulate gene
expression).
Some proteins can inhibit transcription by blockingpolymerase activity.
Some proteins can act both as repressors and activators
of transcription.
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Transcription: chain initiation
Chain initiation:
RNA polymerase locally denatures the DNA. The first base of the new RNA strand is placed
complementary to the +1 site.
RNA polymerase does not require a primer.
The first 8 or 9 bases of the transcript are linked.Transcription factors are released, and the polymerase
leaves the promoter region.
Figure of bacterial transcription initiation.
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.figgrp.2615http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.figgrp.844http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.figgrp.844http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.figgrp.2615 -
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Transcription: chain elongation
Chainelongation:
RNA polymerase moves along the transcribed or templateDNA strand.
The new RNA molecule (primary transcript) forms a short
RNA-DNA hybrid molecule with the DNA template.
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.figgrp.3968http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.figgrp.3968http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.figgrp.3968http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.figgrp.3968 -
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Transcription: chain termination
Most known about bacterial chain termination
Termination is signaled by a sequence that can form ahairpin loop.
The polymerase and the new RNA molecule are released
upon formation of the loop.
Review the transcription animation.
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Transcription: mRNA synthesis/processing
Prokaryotes: mRNA transcribed directly from DNA
template and used immediately in protein synthesis Eukaryotes: primary transcript must be processedto
produce the mRNA
Noncoding sequences (introns) are removed
Coding sequences (exons) spliced together
5-methylguanosine cap added
3-polyadenosine tail added
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Transcription: mRNA synthesis/processing
Removal of introns and splicing of exons can occur
several ways For introns within a nuclear transcript, a spliceosomeis required.
Splicesomes protein and small nuclear RNA (snRNA)
Specificity of splicing comes from the snRNA, some of which contain
sequences complementary to the splice junctions between introns and
exonsAlternative splicingcan produce different forms of a protein from
the same gene
Mutationsat the splice sites can cause disease
Thalassemia Breast cancer(BRCA 1)
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&db=books&doptcmdl=GenBookHL&term=spliceosome+AND+mboc4%5Bbook%5D+AND+372675%5Buid%5D&rid=mboc4.figgrp.1020http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mboc4.figgrp.1368http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&db=books&doptcmdl=GenBookHL&term=spliceosome+AND+hmg%5Bbook%5D+AND+226630%5Buid%5D&rid=hmg.figgrp.1124http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.figgrp.4004http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=113705http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=113705http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.figgrp.4004http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&db=books&doptcmdl=GenBookHL&term=spliceosome+AND+hmg%5Bbook%5D+AND+226630%5Buid%5D&rid=hmg.figgrp.1124http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mboc4.figgrp.1368http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Search&db=books&doptcmdl=GenBookHL&term=spliceosome+AND+mboc4%5Bbook%5D+AND+372675%5Buid%5D&rid=mboc4.figgrp.1020 -
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Transcription: mRNA synthesis/processing
RNA splicing inside the nucleus on particles called
spliceosomes. Splicesomes are composed of proteins and small RNA
molecules (100200 bp; snRNA).
Both proteins and RNA are required, but some suggesting
that RNA can catalyze the splicing reaction. Self-splicing in Tetrahymena: the RNA catalyzes its own
splicing
Catalytic RNA: ribozymes
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From gene to protein: genetic code
Central Dogma
Information travels from DNA to RNA to Protein Is there a one-to-one correspondence between DNA, RNA and Protein?
DNA and RNA each have four nucleotides that can form them; so yes, there
is a one-to-one correspondence between DNA and RNA.
Proteins can be composed of a potential 20 amino acids; only four RNA
nucleotides: no one-to-one correspondence.
How then does RNA direct the order and number of amino acids in a protein?
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From gene to protein: genetic code
How many bases are required for each amino acid?
(4 bases)
2bases/aa
= 16 amino acidsnot enough (4 bases)3bases/aa= 64 amino acid possibilities
Minimum of 3 bases/aa required
What is the nature of the code?
Does it have punctuation? Is it overlapping?
Crick, F.H. et al. (1961) Nature192, 122732.
(http://profiles.nlm.nih.gov/SC/B/C/B/J/)
3-base, nonoverlapping code that is read from a fixed point.
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From gene to protein: genetic code
Nirenberg and Matthaei: in vitro protein translation
Found that adding rRNA prolonged cell-free protein synthesisAdding artificial RNA synthesized by polynucleotide phosphorylase
(no template, UUUUUUUUU) stimulated protein synthesis more
The protein that came out of this reaction was polyphenylalanine
(UUU = Phe)
Other artificial RNAs: AAA = Lys; CCC =Pro
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From gene to protein: genetic code
Nirenberg:
Triplet binding assay: add triplet RNA, ribosomes, binding factors,GTP, and radiolabeled charged tRNA (figure)
UUU trinucleotide binds to Phe-tRNA
UGU trinucleotide binds to CYS-tRNA
By fits and starts the triplet genetic codewas worked out.
Each three-letter word (codon) specifies an amino acid ordirections to stop translation.
The code is redundant or degenerate: more than one way to
encode an amino acid
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From gene to protein: Translation
Components required for translation:
mRNA Ribosomes
tRNA
Aminoacyl tRNA synthetases
Initiation, elongation and termination factors
Animation of translation
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Translation: initiation
Ribosome small subunit binds to mRNA
Charged tRNA anticodon forms base pairs with the mRNAcodon
Small subunit interacts with initiation factors and special
initiator tRNA that is charged with methionine
mRNA-small subunit-tRNA complex recruits the largesubunit
Eukaryoticand prokaryoticinitiation differ slightly
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Translation: initiation
The large subunit of the ribosome contains three binding
sitesAmino acyl (A site)
Peptidyl (P site)
Exit (E site)
At initiation,
The tRNAfMetoccupies the P site
A second, charged tRNA complementary to the next codon binds
the A site.
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Translation: elongation
Elongation
Ribosome translocates by three bases after peptide bondformed
New charged tRNA aligns in the A site
Peptide bondbetween amino acids in A and P sites is
formed Ribosome translocates by three more bases
The uncharged tRNA in the A site is moved to the E site.
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Translation: elongation
EF-Tu recruits charged tRNA to A site. Requires
hydrolysis of GTP Peptidyl transferase catalyzes peptide bond formation
(bond between aa and tRNA in the P site converted to
peptide bond between the two amino acids)
Peptide bond formation requires RNA and may be aribozyme-catalyzed reaction
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Translation: termination
Termination
Elongation proceeds until STOP codon reached UAA, UAG, UGA
No tRNA normally exists that can form base pairing with a
STOP codon; recognized by a release factor
tRNA charged with last amino acid will remain at P site Release factors cleave the amino acid from the tRNA
Ribosome subunits dissociate from each other
Review the animation of translation
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