Noor RNA

Abu Farsakh
Structure
Nabeel
DeebTranscription
20
9
, Basma
&
Basheer
20/10/201
, Rasha Al-Ebbini
0

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Genetics – Lecture 9
Wednesday 20-10-2010
Done By: Noor Abu Farsakh, Basma Deeb, Rasha Al-
Ebbini

RNA Structure & Transcription

As we know, concerning the DNA and RNA in terms of nitrogen

bases that the only difference between them is that thymine is
present in DNA and that uracil is in RNA, and the difference
between thymine and uracil is the methyl group in thymine.

As you know, RNA in terms of its primary structure is single

stranded, and generally speaking there are four major
ribonucleotides, but sometimes you will see unusual nitrogen
bases within them that they were not coded but were extensively
modified posttranscriptionally, and here are some examples
on them:
Dihydrouridine, Pseudouridine, 1-methylguanosine, 7-
methylguanosine, 1-methyladenosine, 2-thiocytidine, 5-
methylcytidine and Ribothymine (it is unusual to see thymine in
RNA).

Q: Why do we have modified nitrogen bases?

These modifications will give the proper tertiary structure for the
RNA to be functional.

RNA monomers are ribonucleoside monophosphates that are

inserted from ribonucleoside triphosphate –similar to the DNA
synthesis- so a ribonucleoside monophosphate is inserted and a
pyrophosphate is released, and a 3’-5’ phosphodiester bond is
being formed. The RNA being synthesized has a 3’ end and a 5’
end just like the single stranded DNA.

There are different classes of RNA molecules in

prokaryotes:
1- Transfer RNA
2- Ribosomal RNA

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3- Messenger RNA

Transfer RNA:
The transfer RNA has a secondary structure and a tertiary
structure that is proper for its function of protein biosynthesis and
carrying the amino acids. It is a substrate for transfer RNA amino
acyl-transferase. And it has a certain structure and specific sites
in order to carry those amino acids specifically to do its function.

The anticodon loop region in tRNA is responsible for the codon-

anticodon interaction between the transfer RNA and the
messenger RNA; this leads to the insertion of the amino acid to
the growing chain of the polypeptide.

The Acceptor region is always ACC in tRNA in order to carry the

proper amino acid.

Every amino acid has at least one special tRNA for itself. In
nature we have about 70 different tRNA types. Although their
sequences are homologous, there must be some differences
between different types of tRNA for different amino acids, and
that is because of the different modifications in their nitrogen
bases.

Ribosomal RNA:
It is a major component of ribosomes and it is important in protein
biosynthesis. Ribosomes are nucleoproteins (nucleic acids and
proteins mixed together), and it is composed of two subunits; one
large subunit and other small subunit in both eukaryotes and
prokaryotes.

In prokaryotes the 16S ribosomal RNA is a major component of

the small subunit of the ribosome. While the 23S and the 5S are
the two major RNA classes that compose the large ribosomal
subunit.

Q: What do we mean by the 16S, 23S, and 5S?

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This is a measure of the size, and the “S” stands for a specific unit
called “Svedberg unit”. Upon fractionation of the different species
of RNA using cesium chloride gradient centrifugation, they will
sediment at different positions in the tube of cesium chloride
gradient, depending on their densities and their sizes. So from
this approach, they are named in this way.

Messenger RNA:
It will carry the information from the gene (DNA) by transcription,
then its sequence will be translated into protein to give the
specific phenotype.

As you can see in the figure above, mRNA has a 5’ region and a 3’
region. At the 5’ region, there is a sequence of uridine nitrogen
bases. This sequence of uridines or pyrimidines is important
because it will be the recognition site for rRNA 16S (the small
ribosomal subunit). mRNA has a sequence called the “Shine-
Dalgarno sequence” -named after the scientist who discovered it-
that is used in order to help the purine sequence in the small
ribosomal subunit (16S) to bind to the messenger RNA at this
region to make the complex to initiate translation and protein
synthesis.

In the figure (to the right of Shine-Dalgarno sequence) there is a

region that will not be translated, and here is where the ribosome
will find the mRNA by looking at this sequence via the Shine-
Dalgarno sequence.

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Then the ribosome will look for the initiation codon in the mRNA
which is “AUG”, and from this codon translation starts. So
theoretically speaking, all prokaryotic proteins will begin with AUG
in terms of mRNA, and methionine in terms of amino acids.

Then the ribosome will extend for the whole translated region.

Now look at the “AAU” codon. It is called the termination codon

(stop codon). Once the ribosome reaches here, the translation
process will be stopped.

* Note that the AAU codon does not code for any amino acid.
* Also note that mRNA in prokaryotes has exons only; there are no
introns.
The Process of Transcription
It can be classified into three major stages:
1- Initiation stage
2- Elongation stage
3- Termination stage

Initiation Stage:
Once RNA polymerase binds after a lot of effort by the help of a
lot of accessory proteins, it will first form the closed promoter
complex. And by the use of other accessory protein that help the
RNA polymerase, the double stranded DNA will open, and the
open promoter complex will form. Then, the first nucleotide will
be synthesized according to the DNA template.

So these are the initiation steps: formation of a closed complex,

formation of an open complex, and formation of the first
ribonucleotide. Once that happens, the second stage of
transcription will take place, which is the elongation.

* Remember: Transcription of RNA does NOT require a primer;

RNA polymerase doesn’t need a primer.

* Also note that in each step, RNA polymerase will change its
tertiary structure to do the required function.

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Q: Is the sequence for replication the same as the
initiation sequence of transcription?
The answer is No. The sequence that initiates transcription is the
sequence of the promoter and the 5’ flanking region of the
promoter, which is different from the origin of replication in DNA
replication.

Q: Is every single DNA strand used in the transcription?

No, the transcription process goes from the 5’ to the 3’ direction
-like DNA replication- so RNA polymerase must use the DNA
strand that is 3’-5’ direction as a template (the coding strand).
The other strand which is 5’-3’ direction is not used in
transcription (the non-coding strand).

Q: Does RNA polymerase differentiate between the

dominant genes and the recessive (mutant) genes?
No, RNA polymerase will transcribe both the recessive and
dominant genes. But when it is translated by tRNA, and reaches
to the non-sense codon (the stop codon, which is a premature
terminator; not the original terminator), it will not code for any
amino acid, and the protein will be defected.

Q: As you said, the coding strand is a DNA strand that

goes from the 3`- 5` direction, why can't I use the other
DNA strand (the non-coding strand) in the other direction?
This doesn’t happen in nature; the RNA polymerase doesn’t use
non-coding strands; it only uses the coding strand as a template.

Q: Is the direction of the coding strand from 5`-3` or 3`-5`?

Transcription is from 5`- 3` regarding the newly synthesized
mRNA, so the template should be 3`-5` in the DNA coding strand.

Q: Is the synthesis of mRNA unidirectional?

Yes, it is unidirectional.

Q: Why do we have double-stranded DNA, if we only use

one of the strands as a template in the transcription?

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For DNA replication! If we don’t have double stranded DNA, we
will not be able to transmit our genetic information from one
generation to the other.
For transcription, only one strand will be functional. The mRNA
sequence will be exactly the same as the sequence of the non-
coding strand, and it will be complementary to the coding strand
template.

Promoter Structure in Prokaryotes

What you see here is a segment of DNA that represents a specific

gene. The gene will start from the nucleotide labeled as +1, then
goes down stream (to the right). In the 5` flanking region of the
gene (the upstream region), there are sequences called the
promoter. So, the promoter is a specific sequence that is linked
directly to the 5` region of the gene.

If you look in some details regarding the promoter, you'll see

numbers: -10, -30, -200, -1000. Promoters, in size, are different
from one gene to another. They have some sequences which are
very important to the gene. Every gene must have a promoter;

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without the promoter the gene will not be expressed; it will not
give an mRNA nor proteins.

The -10 region has this type of sequence: TATAAT. And -30 region
has this sequence: TTGACA. If you look at different promoters of
different genes in prokaryotics, you'll see some of these are the
same in all promoters. The -10 region sequence is called the
pribnow box (found in all promoters).

A mutation in A (95%), as an important base in the promoter of

every gene, destroys the promoter so there won't be any gene
expression. But if there is a mutation in T (44%), the promoter
efficiency could be lower, but it will not be blocked.

So, there are sub sequences in the promoter that are important
for the expression of any gene. -10 region (the pribnow box
region) is very important, because it will be used as a recognition
sequence for specific proteins including RNA polymerase. RNA
polymerase won't bind to any other place of the gene by its
accessory proteins. These proteins will seek randomly all the gene
till they find the pribnow box and the -30 region; they will bind
there and give a signal to the RNA polymerase to come and bind
here. So this is the importance of the -10 & -30 regions.

* T (96%) means >> this T is found at position (-7) in 96% of

different prokaryotic promoter genes.

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* If you induce mutation in the promoter gene by directing the
mutation to this T, you can kill the organism; because the
promoter is destroyed >> no gene expression.

Prokaryotic RNA Polymerase Structure

There is only one RNA polymerase enzyme (we know DNA has
more than one; DNA pol I, pol II, & pol III). So, there's only one
RNA polymerase for transfer RNA, ribosomal RNA, or messenger
RNA.

Remember that tRNA has its gene & rRNA has its gene, and these
are transcribed by RNA polymerase.

RNA polymerase is a multi-subunit enzyme:

α subunit: its function is still unkown.
β subunit: forms phosphodiester bonds (5'-3' polymerizing
activity) *very important*
β' subunit: binds to the template
δ subunit: important for promoter recognition & transcription
initiation

It was found that the β subunit, which is the principal subunit in

RNA polymerase, is inhibited by Rifampicin (an antibiotic). This
antibiotic is used in treatment of tuberculosis. It inhibits the
activity of β subunit while the other subunits aren't affected.

When these subunits are together, the RNA polymerase is called

Holoenzyme. Once δ factor dissociates from the holoenzyme,
we'll end up with the Core Polymerase (αββ').

To initiate transcription, the holoenzyme is needed. For

elongation, the core enzyme is needed. So the function of δ is to
initiate transcription.
Look at these figures:
Ka Values (M^-1)
Any DNA Promoter DNA
(nonspecific) (specific)

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 Core Enzyme 2 x 10^11
Holoenzyme 10^7 10^13 to 10^15
* Ka: the association constant; as it increases, the binding affinity
increases.

We can conclude that:

- The binding affinity of the holoenzyme to "any" DNA
segment is less than that of the core enzyme.
- The binding affinity of the holoenzyme to the "promoter"
DNA is much greater than its binding affinity to "any" DNA
segment; due to the presence of δ.
- δ destabilizes nonspecific binding to non-promoter DNA.
- δ stabilizes specific binding to promoter DNA; this
accelerates the search for promoter DNA.

Q: Why does this happen? What is the effect of δ?

δ has a specific sequence in its tertiary structure that helps
recognize the pribnow box. Without δ, the core enzyme won't be
able to recognize the pribnow box.

So, what is the function of δ?

It accelerates initiation of transcription by helping RNA
polymerase to bind to the promoter. After that, δ will leave! If δ
doesn't leave, it will keep the RNA polymerase associated with the
promoter, so it won't be able to polymerize and synthesize RNA.
Mechanism of RNA synthesis
A DNA strand is used as a template, and ribonucleosides
triphosphotase are used as substrates for RNA polymerase, which
catalyses the formation of 3'-5' phosphodiester bonds while
removing pyrophosphate (PPi).

The OH present on C2 (in the ribose sugar) will affect the

phosphodiester bond & destabilize it. This OH isn't found in DNA,
which makes its structure highly stable. DNA structure should be
highly stable because it contains the genes. In contrast, the turn
over number of RNA is high; we don't need to store RNA; it is
synthesized when it is required. It's very important that the RNA
won't be stored forever (it has a short half life), because RNA
polymerase doesn't have proofreading activity; so by time it can

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be changed (although, the rate of making mistakes in RNA is very
very low). So, RNA is degraded & synthesized again when needed.

Eukaryotic Transcription
Classes of Eukaryotic Cellular RNAs:
There are 6 different classes:

1. Ribosomal RNA (rRNA): important in protein biosynthesis. It

is made of a large subunit & a small subunit:
- 18S (small subunit)
- 28S (large subunit)
- 5.8S (large subunit)
- 5S (large subunit)

2. Transfer RNA (tRNA): important in translation.

3. Messenger RNA (mRNA): important in transcription into

proteins.

4. Heterogeneous Nuclear RNA (hnRNA): a group of mRNAs

of different sizes in the nucleus. They are intermediates in the
process of maturation of mRNA (precursors of mRNA).

5. Small Nuclear RNA (snRNA): important role in splicing of

mRNA.
- U1, U2, U3, U4, U5, U6, U7, U8, U9, U10 ...

6. Small Cytoplasmic RNA (scRNA)

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