Gene expresion transcription.pdf

Gene Expression - Transcription
When a protein is needed by a cell, the genetic code for
that protein must must be read from the DNA and
processed.
A two step process:
1. Transcription = synthesis of a single-stranded RNA
molecule using the DNA template (1 strand of DNA
is transcribed).
2. Translation = conversion of a messenger RNA
sequence into the amino acid sequence of a
polypeptide (i.e., protein synthesis)
✓ Both processes occur throughout the cell cycle.
Genetics from Medel experiments to
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Four different types of RNA, each encoded by different genes:
1. mRNA Messenger RNA, encodes the amino acid sequence
of a polypeptide
2. tRNA Transfer RNA, transports amino acids to ribosomes
during translation
3. rRNA Ribosomal RNA, forms complexes called ribosomes
with protein, the structure on which mRNA is
translated
4. snRNA Small nuclear RNA, forms complexes with proteins
used in eukaryotic RNA processing (e.g., exon
splicing and intron removal).
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Transcription: How is an RNA strand synthesized?
1. Regulated by gene regulatory elements within each gene.
2. DNA unwinds next to a gene.
3. RNA is transcribed 5’ to 3’ from the template (3’ to 5’).
4. Similar to DNA synthesis, except:
✓ NTPs instead of dNTPs (no deoxy-)
✓ No primer
✓ No proofreading
✓ Adds Uracil (U) instead of thymine (T)
✓ RNA polymerase
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Three Steps to Transcription:
1. Initiation
2. Elongation
3. Termination
✓ Occur in both prokaryotes and eukaryotes.
✓ Elongation is conserved in prokaryotes and eukaryotes.
✓ Initiation and termination proceed differently.
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Step 1-Initiation, E. coli model:
Fig. 5.3
Each gene has three regions:
1. 5’ Promoter, attracts RNA polymerase
-10 bp 5’-TATAAT-3’
-35 bp 5’-TTGACA-3’
2. Transcribed sequence, or RNA coding sequence
3. 3’ Terminator, signals the stop point
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Step 1-Initiation
1. RNA polymerase combines with sigma factor (a polypeptide) to
create RNA polymerase holoenzyme
✓ Recognizes promoters and initiates transcription.
✓ Sigma factor required for efficient binding and transcription.
✓ Different sigma factors recognize different promoter
sequences.
2. RNA polymerase holoenzyme binds promoters and untwists DNA
✓ Binds loosely to the -35 promoter (DNA is d.s.)
✓ Binds tightly to the -10 promoter and untwists
3. Different types and levels of sigma factors influence the level and
dynamics of gene expression (how much and efficiency).
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Step 2-Elongation
1. After 8-9 bp of RNA synthesis occurs, sigma factor is released and
recycled for other reactions.
2. RNA polymerase completes the transcription at 30-50 bp/second.
3. DNA untwists rapidly, and re-anneals behind the enzyme.
4. Part of the new RNA strand is hybrid DNA-RNA, but most RNA is
displaced as the helix reforms.
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Step 3-Termination
Two types of terminator sequences occur in prokaryotes:
1. Type I (-independent)
Palindromic, inverse repeat forms a hairpin loop and is believed
to physically destabilize the DNA-RNA hybrid.
2. Type II (-dependent)
Involves  factor proteins, believed to break the hydrogen bonds
between the template DNA and RNA.
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Prokaryotes possess only one type of RNA polymerase
✓ transcribes mRNAs, tRNAs, and rRNAs
Transcription is more complicated in eukaryotes
Eukaryotes possess three RNA polymerases:
1. RNA polymerase I, transcribes three major rRNAs 12S, 18S, 5.8S
2. RNA polymerase II, transcribes mRNAs and some snRNAs
3. RNA polymerase III, transcribes tRNAs, 5S rRNA, and snRNAs
*S values of rRNAs refer to molecular size, as determined in a sucrose gradient
(review box 5.1)
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Sucrose density gradient centrifugation technique for separating and
isolating RNA molecules in a mixture
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Transcription of protein-coding genes by RNA polymerase II
✓ Recall that RNA polymerase II transcribes mRNA
✓ Like prokaryotes, eukaryotes require a promoter, two types
1. Basal elements (near the transcription start, ~-25 bp)
“TATA Box” = TATAAAA
*AT-rich DNA is easier to denature than GC-rich DNA
2. Proximal elements (located upstream, ~-50 to -200 bp)
“Cat Box” = CAAT and “GC Box” GGGCGG
✓ Different combinations occur near different genes
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Transcription of protein-coding genes by RNA polymerase II
✓ Transcription factors (TFs) also are required by RNA polymerases (function is
similar to sigma factor).
✓ TFs are proteins, assembled on basal promoter elements
✓ Each TF works with only one kind of RNA polymerase (required by all 3 RNA
polymerases).
✓ Numbered (i.e., named) to match their RNA polymerase.
TFIID, TFIIB, TFIIF, TFIIE, TFIIH
✓ Binding of TFs and RNA polymerase occurs in a set order in protein coding genes.
✓ Complete complex (RNA polymerase + TFs) is called a pre-initiation complex (PIC).
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Order of binding is: IID + IIB + RNA poly. II + IIF +IIE +IIH
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Binding of Activator Factors
✓ High-level transcription is induced by binding of activator factors to
DNA sequences called enhancers.
✓Single or multiple copies in either orientation
✓Usually located upstream
✓Can be several kb from the gene
✓Silencer elements and repressor factors also exist
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Production of the mRNA molecule
Three main parts:
1. 5’ untranslated region (UTR) leader sequence
2. Coding sequence, specifies amino acids to be translated
3. 3’ untranslated region (UTR) trailer sequence
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mRNA Differences between prokaryotes and eukaryotes:
Prokaryotes
1. mRNA transcript is mature, and used directly for translation
without modification.
2. Since prokaryotes lack a nucleus, mRNA also is translated on
ribosomes before is is transcribed completely (i.e., transcription
and translation are coupled).
3. Prokaryote mRNAs are polycistronic, they contain amino acid
coding information for more than one gene.
Eukaryotes
1. mRNA transcript is not mature (pre-mRNA); must be processed.
2. Transcription and translation are not coupled (mRNA must first
be exported to the cytoplasm before translation occurs).
3. Eukaryote mRNAs are monocistronic, they contain amino acid
sequences for just one gene.
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, Prokaryotes and Eukaryotes
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Production of mature mRNA in eukaryotes:
1. 5’ cap
✓ After 20-30 nucleotides have been synthesized, the 5’-end of
the mRNA is capped 5’ to 5’ with a guanine nucleotide.
✓ Results in the addition of two methyl (CH3) groups.
✓ Essential for the ribosome to bind to the 5’ end of the mRNA.
2. Poly (A) tail,
✓ 50-250 adenine nucleotides are added to 3’ end of mRNA.
✓ Complex enzymatic reaction (illustrated in Fig. 5.10).
✓ Stabilizes the mRNA, and plays an important role in
transcription termination.
3. Introns (non-coding sequences between exons) are removed and
exons (amino acid coding sequences) are spliced.
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Introns and exons:
✓ Eukaryote pre-mRNAs often have intervening introns that must be
removed during RNA processing (as do some viruses).
intron = non-coding DNA sequences between exons in a gene.
exon = expressed DNA sequences in a gene, code for amino acids.
1993: Richard Roberts (New England Biolabs) & Phillip Sharp (MIT)
Fig. 5.11
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The general concept prevailing during the mid 1970s
regarding the hereditary material and its function can
be summarized as follows.
A gene exists as a continuous stretch (segment) within
a long, double-stranded DNA molecule.
When the gene is activated, its information is copied
into a single-stranded RNA molecule, called messenger
RNA, which translates the information into a protein
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This simple picture of the sequence of events radically
changed through the discovery made in 1977
by Richard J. Roberts, working at the Cold Spring
Harbor Laboratory on Long Island, New York,
and Phillip A. Sharp, working at the Massachusetts
Institute of Technology in Cambridge, USA.
They found that an individual gene can comprise not
only one but several DNA segments separated by
irrelevant DNA Such discontinuous genes exist in
organisms more complex than those studied earlier.
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Gene structure and the flow of genetic information
in bacteria (A) and higher organisms (B). In bacteria,
the genetic information is stored as a continuous
segment of DNA, and the messenger RNA can
immediately direct the synthesis of the
corresponding protein. In higher organisms, the gene
is usually split, and the messenger RNA has to be
processed by splicing before it can be translated into
a protein.
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How the discovery was made
Roberts and Sharp were studying the genetic
material in adenovirus,
a virus causing common cold.
This virus infects the cells of higher organisms, and
its genome has many properties resembling those of
the host cell.
At the same time, adenovirus has a simple
structure, making it a very valuable experimental
model for studying genes and their function in
higher organisms.
The genome of adenovirus consists of one single
long DNA molecule. Roberts’ and Sharp’s aim was to
determine where in the genome different genes
were located.
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In biochemical experiments it was shown that one end
of an adenovirus messenger RNA did not behave as
expected.
One of several possible explanations was that the DNA
segment corresponding to this end was not located in
the immediate vicinity of the rest of the gene.
To determine where this segment was located on the
long DNA molecule, they used electron microscopy.
They surprisingly found that a single RNA molecule
corresponded to no less than four well-separated
segments in the DNA molecule
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Roberts and Sharp came to the conclusion that the genetic
information in the gene was discontinuously organized in the
genome, a conclusion that contradicted the commonly held
view regarding the structure of genes.
The discovery immediately led to intensive research to find out
whether this gene structure is present also in other viruses and
in ordinary cells.
Very soon after the initial discovery, several researchers could
show that a discontinuous (or split) gene structure was
common – and in fact the most common gene structure in
higher organisms.
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The importance of the discovery
A gene may thus consist of several
segments, usually termed exons separated
by intervening DNA, termed introns.
This knowledge has radically changed our
view on how the genetic material has
developed during the course of evolution.
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he process is called splicing and in higher organisms it represents an
additional step in the transfer of information as compared to what usually
occurs in lower organisms
The importance of splicing became particularly apparent, when it was
found that it is not always the same segments that are identified as exons
and are included in the final RNA molecule.
In different tissues or developmental stages, the final RNA molecule may
be different due to the utilization of alternative exon combinations.
Thus, the same DNA region can in many cases determine the structure of
many different proteins. The process is called alternative splicing and
represents a fundamentally new principle: the genetic message, which
gives rise to a particular product, is not definitely established at the stage
when the RNA is first synthesized.
Instead, it is the splicing pattern that determines the nature of the final
product.
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The most studied of such diseases is beta-thalassemia, an
anemia prevalent primarily in some Mediterranean countries.
The disease is due to a faulty protein, which forms part of
hemoglobin in red blood cells.
The protein is called beta-globin. If no or badly functioning
beta-globin is made, the life-span of the red blood cells is
shortened resulting in anemia.
In different patients, small defects in the genetic material have
been found, resulting in errors in the splicing process and thus
in the synthesis of poorly functioning beta-globin.
in the upper part of figure 3 the normal splicing of beta-globin
RNA is shown (A).
If the globin gene is damaged (marked by an arrow) it may, for
example, lead to the formation of a larger than normal exon
during splicing (B), or to the formation of a completely new
exon (C). Genetics from Medel experiments to
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Another example showing the connection between disease
and the organization of the genetic material into exons and
introns is chronic myeloic leukemia, a type of cancer of the
blood.
Characteristic for this disease is the presence in tumor cells of
a special, shortened chromosome, called the Philadelphia
chromosome,
named after the city in which it was discovered. This
chromosome has arisen in a white blood cell by fusion of one
end of chromosome 22 to one end of chromosome 9.
At the break-point, a large portion of a cancer gene has been
joined to another gene.
Here we are thus dealing with two genes, which are now
copied into one single RNA molecule. During the splicing
process exons from the two genes are spliced to form an RNA
molecule that specifies the synthesis of a new protein, a so-
called fusion protein.
This new protein gives rise to leukemia.
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mRNA splicing of exons and removal of introns:
1. Introns typically begin with a 5’-GT(U) and end with AG-3’.
2. Cleavage occurs first at the 5’ end of intron 1 (between 2 exons).
3. The now free G joins with an A at a specific branch point sequence in the
middle of the intron, using a 2’ to 5’ phosphodiester bond.
✓ Intron forms a lariat-shaped structure.
4. Lariat is excised, and the exons are joined to form a spliced mRNA.
5. Splicing is mediated by splicosomes, complexes of small nuclear RNAs
(snRNAs) and proteins, that cleave the intron at the 3’ end and join the
exons.
6. Introns are degraded by the cell.
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Post-transcriptional modification, mRNA editing:
1. Adds or deletes nucleotides from a pre-RNA, or
chemically alters the bases, so the mRNA bases do not
match the DNA sequence.
2. Can results in the substitution, addition, or deletion of
amino acids (relative to the DNA template).
3. Generally cell or tissue specific.
4. Examples; protozoa, slime molds, plant organelles,
mammals
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Genes that do not code proteins also are transcribed:
1. rRNA, ribosomal RNA
✓ Catalyze protein synthesis by facilitating the binding of
tRNA (and their amino acids) to mRNA.
2. tRNA, transfer RNA
✓ Transport amino acids to mRNA for translation.
3. snRNA, small nuclear RNA
✓ Combine with proteins to form complexes used in RNA
processing (splicosomes used for intron removal).
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1. Synthesis of ribosomal RNA and ribosomes:
1. Cells contain thousands of ribosomes.
2. Consist of two subunits (large and small) in prokaryotes and
eukaryotes, in combination with ribosomal proteins.
3. E. coli 70S model:
✓ 50S subunit = 23S (2,904 nt) + 5S (120 nt) + 34 proteins
✓ 30S subunit = 16S (1,542 nt) + 20 proteins
4. Mammalian 80S model:
✓ 60S subunit = 28S (4,700 nt) +5.8S (156 nt) + 5S (120 nt) +
50 proteins
✓ 40S subunit = 18S (1,900 nt) + 35 proteins
5. DNA regions that code for rRNA are called ribosomal DNA (rDNA).
6. Eukaryotes have many copies of rRNA genes tandemly repeated.
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1. Synthesis of ribosomal RNA and ribosomes:(continued):
7. Transcription occurs by the same mechanism as protein-coding genes,
but generally using RNA polymerase I.
8. rRNA synthesis requires its own array transcription factors (TFs)
9. Coding sequences for RNA subunits within rDNA genes contain internal
(ITS), external (ETS), and nontranscribed spacers (NTS).
10. ITS units separate the RNA subunits through the pre-rRNA stage,
whereupon ITS & ETS are cleaved out and rRNAs are assembled.
11. Subunits of mature ribosomes are bonded together by H-bonds.
12. Finally, transported to the cytoplasm to initiate protein synthesis.
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Mammalian example of 80S rRNA
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2. Synthesis of tRNA:
1. tRNA genes also occur in repeated copies throughout the genome,
and may contain introns.
2. Each tRNA (75-90 nt in length) has a different sequence that
binds a different amino acid.
3. Many tRNAs undergo extensive post-transcription modification,
especially those in the mitochondria and chloroplast.
4. tRNAs form clover-leaf structures, with complementary base-
pairing between regions to form four stems and loops.
5. Loop #2 contains the anti-codon, which recognizes
mRNA codons during translation.
6. Same general mechanism using RNA polymerase III, promoters,
unique TFs, plus posttranscriptional modification from pre-tRNA.
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3. Synthesis of snRNA (small nuclear RNA):
• Form complexes with proteins used in eukaryotic
RNA processing, splicing of mRNA after introns are
removed.
• Transcribed using RNA polymerase II or III.
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