Download 12-Transcription-The Relationship Between Genes and Proteins

12th Week
The Relationship
Transcription Between Genes and
Proteins
Gihan E-H Gawish, MSc, PhD
Ass. Professor
Molecular Genetics and Clinical
Biochemistry
KSU
Table of Contents
• History: linking genes and proteins
• Getting from gene to protein: transcription
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Evidence for mRNA
Overview of transcription
RNA polymerase
Stages of Transcription
• Promoter recognition
• Chain initiation
• Chain elongation
• Chain termination
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mRNA Synthesis/Processing
References
History: linking genes and proteins
• 1900’s 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)?
History: linking genes and proteins
• Beadle and Tatum (1941) PNAS USA 27, 499–506.
• Hypothesis:
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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:
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Isolated ~2,000 strains from single irradiate spores (Neurospora)
that grew on rich but not minimal medium. Examples: defects in B1,
B6 synthesis.
• Conclusion:
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Genes govern the ability to synthesize amino acids, purines and
vitamins.
History: linking genes and proteins
• 1950s: sickle-cell anemia
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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
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Mutations resulted in single amino acid change
Order of mutations in gene same as order of affected amino acids
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)
Transcription: evidence for mRNA
• Brenner, S., Jacob, F. and Meselson, M. (1961) Nature 190,
576–81.
• Question: How do genes work?
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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?
Transcription: evidence for mRNA
• E. coli cells 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 containing
radioactive uracil.
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:
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Ribosomes from phage-infected cells were “heavy”, banding at the
same 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.
Transcription: evidence for mRNA
• Conclusion
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Expression of phage DNA results in new phage-specific RNA
molecules (mRNA)
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These mRNA molecules are temporarily associated with ribosomes
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Ribosomes do not themselves contain the genetic directions for
assembling individual proteins
Transcription: overview
Transcription requires:
• ribonucleoside 5´ triphosphates:
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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
Transcription: overview
• Features of transcription:
• RNA polymerase catalyzes sugar-phosphate bond
between 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.
Transcription: overview
The three-dimensional structures of RNA polymerases from a prokaryote (Thermus
aquaticus) and a eukaryote (Saccharoromyces cerevisiae). The two largest subunits for each
structure are shown in dark red and dark blue. The similarity of these structures reveals
that these enzymes have the same evolutionary origin and have many mechanistic features
in common.
Transcription: overview
• In prokaryotes transcription and translation are coupled.
Proteins are synthesized directly from the primary
transcript as it is made
• In eukaryotes transcription and translation are separated.
Transcription occurs in the nucleus, and translation occurs
in the cytoplasm on ribosomes.
Animation
comparing eukaryotic and prokaryotic transcription
and translation.
Animation
These two processes are closely coupled in prokaryotes, whereas they are spacially and
temporally separate in eukaryotes. (A) In prokaryotes, the primary transcript serves as
mRNA and is used immediately as the template for protein synthesis. (B) In eukaryotes,
mRNA precursors are processed and spliced in the nucleus before being transported to the
cytosol for translation into protein. [After J. Darnell, H. Lodish, and D. Baltimore.
Molecular Cell Biology, 2d ed. (Scientific American Books, 1990), p. 230.]
Transcription: RNA Polymerase
• DNA-dependent
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DNA template, ribonucleoside 5´ triphosphates, and Mg2+
• Synthesizes RNA in 5´ to 3´ direction
• E. coli RNA polymerase consists of 5 subunits
• Eukaryotes have three RNA polymerases
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RNA polymerase II is responsible for transcription of protein-coding
genes and some snRNA molecules
RNA polymerase II has 12 subunits
Requires accessory proteins (transcription factors)
Does not require a primer
Stages of Transcription
• Promoter Recognition
• Chain Initiation
• Chain Elongation
• Chain Termination
Animation
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 12–15 bp segment of the DNA
(open complex).
• The site where the first base is incorporated into the
transcription 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 basal
transcription factors.
promoter recognition
The formation of the active eukaryotic
initiation complex.
The diagrams represent the complexes
formed on the TATA box by the
transcription
factors
and
RNA
polymerase II. (A) The TFIID complex
binds to the TATA box through its TBP
subunit. (B) TFIID is stabilized by
TFIIA. (C) TFIIB and TFIIH join the
complex on the TATA box while TFIIE
and TFIIF associate with RNA
polymerase II. (D) RNA polymerase is
positioned by TFIIB, and its carboxyterminal domain (CTD) is bound by
TFIID. (E) The CTD is phosphorylated
by TFIIH and is released by TFIID. The
RNA polymerase II is now competent to
transcribe mRNA from the gene.
Promoter recognition: promoter sequences
• Promoter sequences vary considerably.
• RNA polymerase binds to different promoters with
different strengths; binding strength relates to the level of
gene expression
• There are some common consensus sequences for
promoters:
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Example: E. coli –35 sequence (found 35 bases 5´ to the start of
transcription)
Example: E. coli TATA box (found 10 bases 5´ to the start of
transcription)
Promoters recognized by E. coli RNA polymerase
Promoter recognition: enhancers
• Eukaryotic genes may also have enhancers.
• Enhancers can be located at great distances from the
gene 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.
Promoter recognition: enhancers
• Modular transcriptional regulatory regions using Pax6 as an activator.
(A) Promoter and enhancer of the chick lens δ1 crystallin gene. Pax6
interacts with Sox2 and Maf to activate this gene. (B) Enhancer of the
rat somatostatin gene. Pax6 activates this gene by cooperating with the
Pdx1 transcription factor. (A after Cvekl and Piatigorsky 1996; B after
Andersen et al. 1999.)
Promoter recognition: other players
• Many proteins can regulate gene expression by
modulating the strength of interaction between the
promoter and RNA polymerase.
• Some proteins can activate transcription (upregulate gene
expression).
• Some proteins can inhibit transcription by blocking
polymerase activity.
• Some proteins can act both as repressors and activators
of transcription.
Transcription: chain initiation
• Chain initiation click-:
• 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 click-.
Stepwise assembly of a transcription-initiation
complex from isolated RNA polymerase II (Pol II)
and general transcription factors
 Once the complete transcription-initiation
complex has assembled, separation of the
DNA strands at the start site to form an opencomplex requires ATP hydrolysis.
 As transcription initiates and the polymerase
transcribes away from the promoter, the CTD
becomes phosphorylated and the general
transcription factors dissociate from the TBPpromoter complex.
 Numerous
other
proteins
transcription initiation in vivo.
participate
in
Transcription of DNA into RNA is
catalyzed by RNA polymerase,
which can initiate the synthesis
of strands de novo on DNA
templates
The nucleotide at the 5′ end of an
RNA strand retains all three of its
phosphate groups; all subsequent
nucleotides release pyrophosphate
(PPi) when added to the chain and
retain only their α phosphate (red).
The released PPi is subsequently
hydrolyzed by pyrophosphatase to
Pi, driving the equilibrium of the
overall
reaction
toward
chain
elongation. In most cases, only one
DNA strand is transcribed into RNA.
Transcription: chain elongation
• Chain elongation:
• RNA polymerase moves along the transcribed or template
DNA strand.
• The new RNA molecule (primary transcript) forms a short
RNA-DNA hybrid molecule with the DNA template.
Transcription: chain termination
• Most known about bacterial
chain termination
• Termination is signaled by
a sequence that can form a
hairpin loop.
• The polymerase and the
new RNA molecule are
released upon formation of
the loop.
• Review the transcription
animation.
Transcription: mRNA synthesis/processing
• Prokaryotes: mRNA transcribed directly from DNA
template and used immediately in protein synthesis
• Eukaryotes: primary transcript must be processed to
produce the mRNA
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Noncoding sequences (introns) are removed
Coding sequences (exons) spliced together
5´-methylguanosine cap added
3´-polyadenosine tail added
Animation
Overview of mRNA processing in eukaryotes
Shortly after RNA polymerase II initiates transcription at the first nucleotide of the first
exon of a gene, the 5′ end of the nascent RNA is capped with 7-methylguanylate.
Transcription by RNA polymerase II terminates at any one of multiple termination sites
downstream from the poly(A) site, which is located at the 3′ end of the final exon. After the
primary transcript is cleaved at the poly(A) site, a string of adenine (A) residues is added.
The poly(A) tail contains ≈250 A residues in mammals, ≈150 in insects, and ≈100 in yeasts.
For short primary transcripts with few introns, polyadenylation, cleavage, and splicing
usually follows termination, as shown. For large genes with multiple introns, introns often
are spliced out of the nascent RNA before transcription of the gene is complete. Note that
the 5′ cap is retained in mature mRNAs.
Transcription: mRNA synthesis/processing
• Removal of introns and splicing of exons can occur
several ways
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For introns within a nuclear transcript, a spliceosome is 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
exons
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Alternative splicing can produce different forms of a protein from
the same gene
Mutations at the splice sites can cause disease
• Thalassemia
• Breast cancer (BRCA 1)
Transcription: mRNA synthesis/processing
• RNA splicing inside the nucleus on particles called
spliceosomes.
• Splicesomes are composed of proteins and small RNA
molecules (100–200 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
Animation