Notes on Transcription and Translation-1

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Notes on Replication,
Transcription and Translation
By
Allen Johnny Borlay (MSc)
Instructor, BIOLOGY 305 Section 3 & 4
Transcription overview
 The first stage in
the expression of genetic information is
transcription of the information in the base sequence of a ds
DNA to form the base sequence of a ss RNA.
 For any particular gene, only one strand of the DNA molecule,
called the template strand, is copied by RNA polymerase.
 Because RNA polymerase moves in the 3' to 5' direction along
the template strand of DNA, the RNA product is antiparallel
and complementary to the template.
 RNA polymerase recognizes start signals (promoters) and
stop signals (terminators) for each of the thousands of
transcription units in the genome of an organism.
 Involves three processes:
 Initiation
Elongation
 Termination

Initiation
Locate the initiation point and unwind the DNA
double helix
A group of proteins called initiation factors move
along the DNA until it reach the promoter region 
special region of nucleotides that signal RNA
polymerase to join the protein complex and begin
synthesis of the RNA strand
The promoter region usually does not vary too much
from organism to organism
The most common sequence of DNA nucleotides
found on the promoter region is called the
consensus sequence
Elongation
Once RNA polymerase unwinds the DNA, it
creates a bubble that exposes the singlestrands of the DNA
Transcription bubble moves down DNA at
constant rate leaving growing RNA strands
protruding from the bubble
Only one of these DNA strands (template or
antisense strand) is actually used to
synthesize the RNA molecule
Hydrogen bonds are created between the
bases, phosphodiester bonds between the
ribonucleotides.
The other stand that is not used is called
sense or coding strand
During transcription, the bubble is maintained
within bacterial RNA polymerase, which
unwinds and rewinds DNA, maintains the
conditions of the partner and template DNA
strands, and synthesizes RNA.
Termination
Here sequence of nucleotides terminate transcription and this
sequence is known as the termination sequence.
When RNA polymerase reaches this termination sequence, a special
protein assist in the dissociation process
Stop sequences at the end of the gene cause phosphodiester bond
formation to cease, transcription bubble to dissociate, and RNA
polymerase to release DNA
The product is immature RNA or pre mRNA (Primary transcript)
The primary product of RNA transcription; the hnRNAs contain both
intronic and exonic sequences.
These hnRNAs are processed in the nucleus to give mature mRNAs
that are transported to the cytoplasm where to participate in protein
synthesis
Eukaryotic transcription differs from prokaryotic
transcription:
three RNA polymerase enzymes
initiation complex forms at promoter
RNAs are modified after transcription
The prokaryotic RNA-pol can bind to the DNA template directly in the
transcription process.
The eukaryotic RNA-pol requires co-factors to bind to the DNA template
together in the transcription process
Eukaryotes Transcription
Regulation very complex
Three different pols
Distinguished by -amanitin sensitivity
Pol I—rRNA, least sensitive
Pol II– mRNA, most sensitive
Pol III– tRNA , moderately sensitive
Each polymerase recognizes a distinct promoter
RNA polymerase in Eukaryotes
RNA polymerase I transcribes rRNA
RNA polymerase II transcribes hnRNA
RNA polymerase III transcribes tRNA
and other small RNAs
Eukaryotic Polymerase I Promoters
RNA Polymerase I
Transcribes rRNA
Sequence not well conserved
Two elements
Core element- surrounds the transcription start site (-45
to + 20)
Upstream control element- between -156 and -107
upstream
Spacing affects strength of transcription
Eukaryotic Polymerase II Promoters
Much more complicated
Two parts
Core promoter
Upstream element
Core promoter
TATA box at ~-10 bases
Initiator—on the transcription start site
Downstream element-further downstream
Many natural promoters lack recognizable versions of
one or more of these sequences
RNA pol III
Precursors to tRNAs,5SrRNA, other small RNAs
Promoter Type I
Lies completely within the transcribed region
5SrRNA promoter split into 3 parts
tRNA promoters split into two parts
Polymerase II-like promoters
EX. snRNA
Lack internal promoter
Resembles pol II promoter in both sequence and position
Post-transcriptional processing of mRNA
The mRNA that id formed directly after transcription is
known as the precursor mRNA/pre-mRNA.
This pre-mRNA has not yet undergone the necessary
modification processes.
In the nucleus, before the RNA can enter the cytoplasm, the
pre-mRNA must undergo three post-transcriptional
modifications.
1. 5’ Guanosine Triphosphate Cap
2.Polyadenylation of the 3’ end and
3. Splicing of Exons
5’ Guanosine Triphosphate Cap
In the nucleus of the cell, the 5’ end of the pre-mRNA is
altered by the attachment of a guanosine nucleotide via a
5’-5’ triphosphate linkage. The guanosine nucleotide is
quickly methylated at the 7th position to form the 7-methyl
guanosine
Why attach the 5’-guanosine cap?
One important of this cap is to protect the mRNA from
degradation during protein synthesis.
It also stabilizes the mRNA and aids in transport across the
nuclear membrane.
Polyadenylation of the 3’ end
In this type of post-transcriptional modification, the
3’-end of the pre-mRNA is removed and a series of
adenosine nucleotides are added.
Therefore, the 3’-end tail that contains the many
adenines is called the polyadenine tail
Just as the 5’cap adds stability and provides
resistance to degradation, the poly-(A) tail also
provide the mRNA with stability and keeps the tail
from degradation
Splicing of Exons
Not all regions of the pre-mRNA molecule code for
proteins
Those regions that do code for proteins are called
exons while those that do not code for proteins are
called introns
Before the pre-mRNA exits the nucleus , special
proteins/enzymes will cut out the introns and
splice together the exons
TRANSLATION
Conversion of a messenger RNA sequence into the amino acid sequence of a
polypeptide (i.e., protein synthesis)
Translation occurs in the cytoplasm where ribosomes are located
mRNA carries the genetic code to cytoplasm, where it is translate by ribosomes.
Ribosomal RNA forms an integral part of the ribosome, without which
translation would not be possible.
Transfer RNA collects the proper amino acids and bring them to the ribosomes
where they are use to synthesize the polypeptide chain
Three step involve: Initiation, Elongation and Termination
Ribosomes
Translation involves special cell machinery known as ribosomes. A given ribosome
consist of two subunit, the small and large subunit
These subunits consist of a combination of ribosomal RNA and proteins.
Ribosomal RNA is synthesized in the nucleolus and travel to the cytoplasm, where
they combine with the mRNA to form ribosomes
Prokaryotes and Eukaryotes have ribosomes that are compose of slightly different
subunits:
Small subunit
Large subunit
Combine
Eukaryotes
40S
60S
80S
Prokaryotes
30S
50S
70S
S stand for Svebery unit, which are the units that measure the rate at which a
given particle or molecule sediments
The svebery units usually refers to how something travel down a test tube when
it is rotating with some angular velocity
Ribosomes cont.
S stand for Svebery unit, which are the units that measure
the rate at which a given particle or molecule sediments
The svebery units usually refers to how something travel
down a test tube when it is rotating with some angular
velocity
The small and large subunits only come together during
the process of translation.
The purpose of ribosome is to use the genetic code to
translate the sequence of nucleotide on the mRNA into
correct sequence of amino acids, thereby synthesizing
the protein
Translation _ Initiation
Once the pre-mRNA undergoes the proper modification in the nucleus,
it becomes mRNA. mRNA then travels through the nuclear pores and
into the cytoplasm of the cell.
Once inside the cytoplasm, with the help from proteins called initiation
factors, the small subunit binds to 5’ end of the mRNA. The small
subunit slides along with mRNA until it reaches the nucleotide
sequence 5’—AUG—3’. This the start codon and it signals for tRNA to
bring the amino acid methionine
Once the tRNA with the methionine binds to the start codon, the large
subunit binds to the small subunit thereby completing the ribosome.
During initiation, the mRNA locate the small ribosomal subunit and bind
to it with the help of protein called initiation factors. The small subunit
move along the mRNA until it reaches the start codon.
Translation _ Initiation cont.
This signals a transfer RNA molecular to find the methionine amino
acid attach to it forming the aminoacyl transfer RNA complex
This complex then binds to the start codon which signals the large
ribosomal subunit to combine with the small subunit, thereby forming
the ribosome
The structure of tRNA is especially suitable for it function- towards the
middle of the RNA strand is a sequence of nucleotides that is
complementary to the start codon sequence; this is known as
anticodon of the tRNA
The 3’ end of the tRNA contains nucleotide sequence that binds the
appropriate amino acids. An enzyme called tRNA synthetase catalyzes
the binding of the tRNA to appropriate amino acid
Translation _ Elongation
Once the ribosome complex has been formed and the tRNA carries the
methionine is in the P-site, the ribosome can now begin sliding along the mRNA
The next aminoacyl tRNA that carries the next amino acid is brought into the Asite. After the P and A sites are filled, an enzyme called peptidyl transferase
catalyzes the formation of peptide bond between the amino acids in the P-site
and the A-site. When the peptide bod is formed, the amino acid detaches from
tRNA in the P-site
The ribosome complex moves three nucleotides in the 5’ to 3’ direction along
the mRNA. This move the tRNA in the P-site into E-site and it relocate the tRNA
carrying the growing polypeptide into the P-site
Once the tRNA moves into the E-site, it is then expelled out of the ribosome
complex. This sliding process is known as translocation
After the A-site is empty, the process can repeat itself to add another amino
acid. This continues until the polypeptide chain is complete
Translation _ Termination
Just like there is a start codon that signals
initiation, there is a stop codon that signals
termination
If the ribosome reads either UAA, UGA or UAG, a
protein called release factor will bind to A-site
This will ultimately cause the polypeptide chain to
break off of the tRNA in the P-site
This then give rise to the ribosome complex to
dissociate and this will end translation.
Post-translation activities
Once the polypeptide is synthesized, it is usually modified before it can be a
mature and active protein. These modifications are called post-translation. Six
types of these modification include
1. Phosphorylation
Certain amino acids, such as serine, threonine and tyrosine found on the
polypeptide chain can be modified via phosphorylation by enzymes called protein
tinases. This type of modification plays a crucial role in the cell cycle and signal
transduction
2. Methylation
Methyl groups (CH3) can be added onto the amino acid by an enzyme called
methyltransferase. Methylation usually increases the hydrophobic character of
the amino acid.
Post-translation activities
3. Glycosylation
This is one of the way in which polypeptides are modified. This process involves
addition sugar components to the proteins. This can affect the proteins
conformation and folding. One example of proteins that are glycosylated are
membrane proteins that act as receptors for important biological molecules.
4. Proteolysis
Certain proteins are synthesized in their inactive (zymogen) form. In order to
activate them, enzymes called proteases must break certain peptide bonds.
Many of the digestive enzymes in the small intestine use this type of posttranslation modification
Post-translation activities cont.
5. N-Acetylation

N-acetylation is the transfer of an acetyl group onto the nitrogen on amino acid.
This process can take place when the ribosome is still translating the
polypeptide chain. In most eukaryotic cells, when translation is still taking place,
the first amino acid (met) is usually removed and replaced by an acetyl group.

N-acetylation plays a crucial role in gene expression. Histones, the protein that
assist in condensing DNA into chromatins can be acetylated, which reduces
their ability to fold and open up the DNA for transcription
6. Lipidation

Lipidation is the process by which lipid components are added onto the
polypeptides. Usually those proteins that are destined to be in membranes, such
as ER, mitochondria or plasma membranes undergo this process.

It increases the proteins affinity to the membranes.
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