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05 Lectuer

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8 views27 pages

05 Lectuer

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Thoria Diab
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© © All Rights Reserved
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Screening

After the transformation step, it is necessary to identify those cells


.that contain plasmid-cloned DNA constructs
Cloning DNA sequences that Encode eukaryotic
proteins
Special strategies are required for cloning and expressing eukaryotic
.coding regions in prokaryotic cells

An eukaryotic structural gene will not function in prokaryotic


organism because there is no mechanism for removing introns from
.transcribed RNA

What is the difference between the prokaryotic and eukaryotic


?genes
Fig. 5.8 General structure of mRNA found in both prokaryotic and
eukaryotic cells
Eukaryotes and prokaryotes
produce mRNAs somewhat
differently
Prokaryotes use the RNA transcript as
.mRNA without modification
Transcription and translation are
.coupled in the cytoplasm
.Messages may be polycistronic
Eukaryotes and prokaryotes
produce mRNAs somewhat
differently
b. Eukaryotes modify pre-RNA into mRNA
.by RNA processing
The processed mRNA migrates from
.nucleus to cytoplasm before translation
.Messages are always monocistronic
Fig. 5.9 Processes for synthesis of functional mRNA in prokaryotes and
eukaryotes
and 3’ Modifications ’5
The newly made 5’ end of the mRNA is modified by 5’
.capping
A capping enzyme adds a guanine, usually 7-methyl
.guanosine (m7G), to the 5’ end using a 5’-to-5’ linkage
.Sugars of the 2 adjacent nt are also methylated
The cap is used for ribosome binding to the mRNA
during translation initiation. (Figure 5.10)

台大農藝系 遺傳學 601 20000


Post-transcriptional mRNA modifications

• 5’ capping.
• Methylated
in caps 0, 1
and 2

Methylated
in caps 1
and 2

Methylated
in cap 2
The 3’ end of mRNA is marked by a poly(A) tail
.)Figure 5.11(
Transcription of mRNA continues through the poly(A)a.
.consensus sequence (AAUAAA)
:Proteins bind and cleave RNA. These include
i. CPSF (cleavage and polyadenylation specificity
.factor)
.ii. CstF (cleavage stimulation factor)
.iii. Two cleavage factor proteins (CFI and CFII)
After cleavage, the enzyme poly(A) polymerase (PAP)
adds A nucleotides to the 3’ end of the RNA, using
ATP as a substrate. PAP is bound to CPSF during this
.process
PABII (poly(A) binding protein II) binds the poly(A) taild.
.as it is produced
Fig. 5.11 Diagram of the 3 end formation of mRNA and the addition of the
poly(A) tail
?Why capping

Caps contribute to the stability of mRNAs by protecting their 5’ ends


.from phosphatases and nucleases
Caps enhance the translation of mRNA by eukaryotic protein-
.synthesizing systems. Cap-binding proteins (CBPs)

Poly(A) tail

It is a string of up to 200 adenine residues (polyadenylic acid (poly


(A) tail)) at the 3’ end.

?Importance

The poly (A) tail provides the means for separating the mRNA fraction
of a tissue form the ribosomal RNA (rRNA) and transfer RNA (tRNA).
Olig (dT) cellulose separation of polyadenylated
.mRNA
Production of Mature mRNA
in Eukaryotes
Eukaryotic pre-RNAs often have introns
(intervening sequences) between the
exons (expressed sequences) that are
.removed during RNA processing
Introns were discovered in 1977 by
Richard Roberts, Philip Sharp and
.Susan Berger
Introns
Removal of introns is necessary for mRNA .1
maturation, as hnRNA (Heteronuclear RNA)
.becomes functional mRNA
in Philip leder’s lab (1978) it was shown that the .2
mouse β-globin pre-mRNA (part of the cell’s
hnRNA) is colinear with the gene that encodes it,
while the mature β-globin mRNA is shorter than
.the gene
The missing RNA was an intron that was removed
.during RNA processing
Introns are found in most eukaryotic genes that .3
encode proteins, and have also been found in
.bacteriophage genes
Processing of Pre-mRNA to
Mature mRNA
Events in eukaryotic mRNA .1
production are summarized in Figure
:5.12. They include
a. Transcription of the gene by RNA
.polymerase II
.b. Addition of the 5’ cap
.c. Addition of the poly(A) tail
.d. Splicing to remove introns
Fig. 5.12 General sequence of steps in the formation of eukaryotic mRNA

.Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings
台大農藝系 遺傳學 601 20000
Introns typically begin with 5’-GU, and end with AG-.2
3’, but mRNA splicing signals involve more than just
.these two small sequences
Spliceosomes are small nuclear ribonucleoproteina.
.particles (snRNPs) associated with pre-mRNAs
Spliceosome principal snRNAs are U1, U2, U4, U5,b.
.and U6
. Each snRNA is associated with several proteinsi.
ii. U4 and U6 are part of the same snRNP. Others are
.in their own snRNPs
iii. Each snRNP type is abundant (≧105 copies per
.nucleus)
:The steps of splicing are outlined in Figure 5.13.3
U1 snRNP binds the 5’ splice junction of the intron, as a result of a.
.base-pairing of the U1 snRNA to the intron RNA
U2 snRNP binds the branch-point sequence upstream of theb.
.3’ splice junction
U4/U6 and U5 snRNPs interact, then bind the U1 and U2 snRNPs,c.
.creating a loop in the intron
U4 snRNP dissociates from the complex, forming the actived.
.spliceosome
The spliceosome cleaves the intron at the 5’ splice junction, e.
freeing it from exon 1. The free 5’ end of the intron bonds to a
specific nucleotide (usually A) in the branch-point sequence to
.form an RNA lariat
The spliceosome cleaves the intron at the 3’ junction, liberating f.
the intron lariat. Exons 1 and 2 are ligated, and the snRNPs are
.released
Fig. 5.13 Model for intron removal by the spliceosome
Fig. 5.13 Model for intron removal by the spliceosome
Fig. 5.13 Model for intron removal by the spliceosome
Cloning DNA sequences that Encode eukaryotic
proteins
Special strategies are required for cloning and expressing eukaryotic
.coding regions in prokaryotic cells

An eukaryotic structural gene will not function in prokaryotic


organism because there is no mechanism for removing introns from
.transcribed RNA

An eukaryotic DNA sequence needs prokaryotic transcriptional and


.translational control sequences to be properly expressed

For eukaryotic genes, the "intorn" problem is overcome by


synthesizing DNA copies of functional mRNA molecules
(complementary DNA (cDNA)) that lack introns and creating tissue-
specific cDNA libraries.
Synthesis of cDNA
Before the mRNA molecules can be cloned into a vector, they must
.be converted to double-stranded DNA

1) Unattached sequences of oligo (dT) molecules are added to the


sample, along with the enzyme reverse transcriptase and the four
dNTPs.
Reverse transcriptase
It is produced by certain retroviruses.

It uses an RNA strand as a template while directing deoxyribonucleotid


into the growing chain.

Thus, when an A, G, C, or U nucleotide of the template RNA strand is


encountered, the complementary deoxyribonucleotide (i.e. T, C, G, or
T) is incorporated into the growing DNA strand.

Before reverse transcriptase synthesis ceases, the DNA strand


usually turns back on itself, and a few nucleotides are added to form
a hairpin loop.
The second DNA strand is synthesized by the addition of
the klenow fragment of E. coli DNA polymerase I.

It is a proteolytic product of E. coli DNA polymerase I that


has both polymerase and 3' exonuclease activities and no
5' exonuclease activity because fractionation of the
digestion products removes the fragment with the 5'
exonuclease activity.

A klenow fragment with only DNA polymerase activity due


to a mutation in the 3' exonuclease sequence is also
available.
It uses the first DNA strand as a template and adds
deoxyribonucleotides to the growing strand, starting from
the 3' hydroxyl group at the end of the hairpin loop.
After the reaction is complete, the sample is treated with
the enzyme RNase H, which degrades the mRNA
molecules, and with S1 nuclease, with opens the hairpin
loops and degrades single-stranded DNA extensions.

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