Transcript Transcription and Translation

Transcription and Translation
The Relationship
Between Genes and
Proteins
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
Table of Contents (continued)
• Getting from gene to protein: genetic code
• Getting from gene to protein: translation
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Translation Initiation
Translation Elongation
Translation Termination
References
History: linking genes and proteins
• 1900’s Archibald Garrod
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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)
These mRNA molecules are temporarily associated with ribosomes
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
• 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.
• Figure comparing eukaryotic and prokaryotic transcription
and translation.
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
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: 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)
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: 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:
• 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.
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
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
From gene to protein: genetic code
• Central Dogma
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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?
From gene to protein: genetic code
• How many bases are required for each amino acid?
• (4 bases)2bases/aa = 16 amino acids—not enough
• (4 bases)3bases/aa = 64 amino acid possibilities
• Minimum of 3 bases/aa required
• What is the nature of the code?
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Does it have punctuation? Is it overlapping?
Crick, F.H. et al. (1961) Nature 192, 1227–32.
(http://profiles.nlm.nih.gov/SC/B/C/B/J/ )
3-base, nonoverlapping code that is read from a fixed point.
From gene to protein: genetic code
• Nirenberg and Matthaei: in vitro protein translation
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Found that adding rRNA prolonged cell-free protein synthesis
Adding 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
From gene to protein: genetic code
• Nirenberg:
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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
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By fits and starts the triplet genetic code was worked out.
Each three-letter “word” (codon) specifies an amino acid or
directions to stop translation.
The code is redundant or degenerate: more than one way to
encode an amino acid
From gene to protein: Translation
• Components required for translation:
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mRNA
Ribosomes
tRNA
Aminoacyl tRNA synthetases
Initiation, elongation and termination factors
• Animation of translation
Translation: initiation
• Ribosome small subunit binds to mRNA
• Charged tRNA anticodon forms base pairs with the mRNA
codon
• Small subunit interacts with initiation factors and special
initiator tRNA that is charged with methionine
• mRNA-small subunit-tRNA complex recruits the large
subunit
• Eukaryotic and prokaryotic initiation differ slightly
Translation: initiation
• The large subunit of the ribosome contains three binding
sites
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Amino acyl (A site)
Peptidyl (P site)
Exit (E site)
• At initiation,
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The tRNAfMet occupies the P site
A second, charged tRNA complementary to the next codon binds
the A site.
Translation: elongation
• Elongation
• Ribosome translocates by three bases after peptide bond
formed
• New charged tRNA aligns in the A site
• Peptide bond between 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.
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 a
ribozyme-catalyzed reaction
Translation: termination
• Termination
• Elongation proceeds until STOP codon reached
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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