Lecture 3
2014
Professor H.D.El-Yassin
1
RNA Synthesis and Processing
Objectives:
1. To understand the transcription process
2. To understand how RNA polymerase generate a new single-stranded RNA
3. To define prmotors and promoter sequence
4. To describe RNA processing eukaryotic cells
Transcription
The process of RNA synthesis directed by a DNA template is termed transcription, and occurs
in three phases: initiation, elongation and termination.
In transcription, DNA is copied to RNA by an enzyme called RNA polymerase .
Transcription to yield an mRNA is the first step of protein biosynthesis .
1. initiation of transcription
i. Promoter sequences. Unlike the initiation of replication, transcriptional initiation does
not require a primer. Promoter sequences are responsible for directing RNA
polymerase to initiate transcription at a particular point. Promoter sequences differ
between prokaryotes and eukaryotes
In genetics, a promoter is a DNA sequence that enables a gene to be transcribed. The
promoter is recognized by RNA polymerase (RNAP), which then initiates transcription.
1. Prokaryotic promoters. The promoters for most prokaryotic genes have three
sequence elements.
a.
Initiation site (startpoint). Transcription for most genes always starts at the
same base (position one). The startpoint is usually purine.
b.
Pribnow box. : lies 9-18 base pairs upstream of the startpoint.
i.
Its either identical to or very similar to the sequence TATAAT.
ii.
The pribnow box also called -10 sequence because it is usually found
10 bp upstream of the startpoint.
c.
The -35 sequence is a component of a typical prokaryotic promoter. It is a
TTGACA. Called -35 sequence because it is usually found 35bp upstream of
the startpoint.
<--upstream
downstream -->
2. Eukaryotic Promoters. Each type of eukaryotic RNA polymerase uses a different
promoter. The promoters used by RNA polymerase I and II are similar to the
prokaryotic promoter in that they are upstream of the startpoint. However, the
promoters used by RNA polymerase III are unique because they are usually
downstream of the startpoint.
Lecture 3
2014
Professor H.D.El-Yassin
2
ii. Initiation factors:
1.
Prokaryotic σ factor is required for accurate initiation of transcription.
2. Eukaryotic initiation factors: the initiation of transcription in eukaryotes is
considerably more complex than in prokaryotes, partly because of the increased
complexity of eukaryotic RNA polymerases and partly because of the diversity of
their promoters.
2. Elongation:
The basic requirement and fundamental mechanism of the elongation phase of RNA synthesis
is the same in prokaryotes and eukaryotes.
1) Template: A single strand of DNA acts as a template to direct the formation of
complementary RNA during transcription.
2) Substrates: the four nucleosides triphosphates are needed as substrates for RNA
synthesis.
3) Direction of synthesis: RNA chain growth proceeds in the 5' to 3' direction.
4) Enzyme:
a. Prokaryotes have a single RNA polymerase responsible for all cellular synthesis.The
structure of RNA polymerase is complex:
b. Eukaryotes have one mitochondrial and three nuclear RNA polymerase. The latter
are distinct enzymes that function to synthesize different RNAs.
3. Termination:
i.
In prokaryotices:
There are two basic classes of termination event in prokaryotes
1. Intrinsic termination (Rho-independent termination) involves terminator sequences within the
RNA as it is being made that signal the RNA polymerase to stop. The terminator sequence
is usually a palindromic DNA sequence that forms a hairpin.
2. Rho-dependent termination uses a termination factor called
ρ factor to stop RNA synthesis
at specific sites. When ρ-factor reaches the RNAP, it causes RNAP to dissociate from the
DNA, terminating transcription.
Lecture 3
2014
Professor H.D.El-Yassin
3
ii. In eukaryotices: Very little is known about how they terminate transcription
Lecture 3
2014
Professor H.D.El-Yassin
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Posttranscriptional RNA processing
Once a gene transcript has been synthesized, numerous post-transcriptional modification or
processing events may be needed before the transcript is functional.
1. Prokaryotes: post-transcriptional processing of RNA is not as extensive in prokaryotes
as in eukaryotes; however, some processing does occur.
2. Eukaryotes: Overall, post-transcriptional processing is more extensive in eukaryotes
than in prokaryotes. This partly is due to the presence of a nucleus from which most
RNAs must be transported. RNAs are processed during this transport. Processing gives
them the characteristics they need to be functional in the cytoplasm such as an
increased stability of mRNAs as well as allowing for another level of gene regulation.
a. The primary transcript (hnRNA) is capped at its 5' end as it is being transcribed.
b. A poly (A) tail, 20 to 200 nucleotide in length is being added to the 3' end of he
transcript.
c. Splicing reactions remove introns and connect the exons.
(The most common cause of
-thalassemia are defects in mRNA splicing of the
-globin gene.
Mutations that affect the splicing create aberrant transcript that are degraded before they are
translated. If patients inherit a single mutant gene thalassemia minor, the disease manifests
itself with a mild anemia. However, patents with homozygous mutations thalassemia major
have sever transfusion-dependent anemia.
Lecture 3
2014
Professor H.D.El-Yassin
5
Lecture 3
2014
Professor H.D.El-Yassin
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Conclusion:
1.
Synthesis of RNA from a DNA template is called transcription
2.
Genes are transcribed by enzymes called RNA polymerases that generate a single-
stranded RNA identical in sequence (with the exception of U in place of T) to one of the
strands of the double-stranded DNA. The DNA strand that directs the sequence of
nucleotides in the RNA by complementary base-pairing is the template strand. The
RNA strand that is initially generated is the primary transcript. The DNA template is
copied in the 3' to 5' direction, and the RNA transcript is synthesized in the 5' to 3'
direction. RNA polymerases differ from DNA polymerases in that they can initiate the
synthesis of new strands in the absence of a primer.
3.
In addition to catalyzing the polymerization of ribonucleotides, RNA polymerases must
be able to recognize the appropriate gene to transcribe, the appropriate strand of the
double-stranded DNA to copy, and the startpoint of transcription . Specific sequences
on DNA, called promoters, determine where the RNA polymerase binds and how
frequently it initiates transcription. Other regulatory sequences, such as promoter-
proximal elements and enhancers, also affect the frequency of transcription.
4.
In bacteria, a single RNA polymerase produces the primary transcript precursors for all
three major classes of RNA: messenger RNA (mRNA), ribosomal RNA (rRNA), and
transfer RNA (tRNA). Because bacteria do not contain nuclei, ribosomes bind to mRNA
as it is being transcribed, and protein synthesis occurs simultaneously with transcription.
Eukaryotic genes are transcribed in the nucleus by three different RNA polymerases,
each principally responsible for one of the major classes of RNA. The primary transcripts
are modified and trimmed to produce the mature RNAs. The precursors of mRNA
(called pre-mRNA) have a guanosine
“cap” added at the 5_-end and a poly(A) “tail” at
the 3_-end. Exons, which contain the coding sequences for the proteins, are separated
in pre-mRNA by introns, regions that have no coding function. During splicing
reactions, introns are removed and the exons connected to form the mature mRNA. In
eukaryotes, tRNA and rRNA precursors are also modified and trimmed, although not as
extensively as pre-mRNA.
Nice to know:
Clinical case:
Anne Niemick is a 4-year-old girl of Mediterranean ancestry whose height and body weight are below
the 20th percentile for girls of her age.
She is listless, tires easily, and complains of loss of appetite and shortnessof breath on exertion. A dull
pain has been present in her right upper quadrant for the last 3 months. Her complexion is slate-gray
and she appears pale. Initial laboratory studies indicate a severe anemia (decreased red blood cell
count) with a hemoglobin of 6.2 g/dL (reference range, 12
–16). A battery of additional hematologic tests
shows that Anne has
+
-thalassemia, intermediate type.
The thalassemias are a heterogenous group of hereditary anemias that constitute the most common
gene disorder in the world, with a carrier rate of almost 7%. The disease was first discovered in countries
around the Mediterranean Sea and was named for the Greek word “thalassa” meaning “sea”. However, it
is also present in areas extending into India and China that are near the equator. The thalassemia
syndromes are caused by mutations that decrease or abolish the synthesis of the
or
chains in the
adult hemoglobin A tetramer. Individual syndromes are named according to the chain whose synthesis is
affected and the severity of the deficiency. Thus, in
° thalassemia, the superscript 0 denotes none of
the
chain is present; in
+
thalassemia, the + denotes a partial reduction in the synthesis of the
chain.
More than 170 different mutations have been identified that cause
thalassemia; most of these interfere
with the transcription of
-globin mRNA or its processing or translation.