RNA structure and functions
Dr. Rana M. W. Hasanato
Assistant Professor & Consultant
Medical Biochemistry department &Clinical Chemistry Unit
College of Medicine and King Khalid University Hospital
---------------------RNA is a polymer composed of alternating units of ribonucleotides connected through
a 3’-5’ phosphodiester bond.
RNA can be single stranded (retroviruses ,HIV) , double stranded (reoviruses) or loop
RNA
STRUCTURE OF RNA:
 Three major types of RNA participate in the process of protein synthesis:
- transfer RNA (tRNA)
- messenger RNA (mRNA)
- ribosomal RNA (rRNA)
• In eukaryotes, small RNA molecules found in the nucleus (snRNA) are
important for the posttranscriptional modifications of mRNA.
 The bases in RNA are:
- adenine (A),
- guanine (G),
- cytosine (C),
- uracil (U).
 Like DNA, these three types of RNA are forms of nucleic acid found in the
cells and unbranched polymeric molecules composed of mononucleotides
joined together by phosphodiester bonds.
 Unlike DNA, They differ as a group from DNA in several ways:
o they are considerably smaller than DNA.
o they contain ribose instead of deoxyribose and uracil instead of thymine.
o most RNAs exist as single stranded entity that are capable of folding
into complex structures.
 The three major types of RNA differ from each other In size, function, and
special structural modifications.
A. Ribosomal RNA:
 rRNA is a type of RNA that is a component of ribosomes and plays a role in the
process of translation (making protein from nucleic acid sequence).
 Ribosomal RNAs (rRNAs) are found in association with several proteins as
components of the ribosomes-the complex structures that serve as the sites for
protein synthesis.
1
 There are three distinct size species of rRNA (23S. 16S. and 5S) in prokaryotic
cells and four rRNA size species (28S. 18S. 5.8S. and 5S) In the eukaryotic
cytosol.
 rRNAs species make up 80% of the total RNA in the cell.
B. Transfer RNA:
 tRNAs are RNA molecules that provide the means of translating the genetic






code.
One end of the tRNA contains a three nucleotide sequence called the anticodon
loop that is complementary to the codon of the mRNA.
The other end of the tRNA is covalently attached to a specific amino acid.
Transfer RNAs (tRNAs), the smallest of the three major species of RNA
molecules (4S), have between 74 and 95 nucleotide residues.
tRNAs species make up about 15 % of the total RNA in the cell.
The tRNA molecules contain unusual bases e.g. dihydrouracil, and have
extensive intrachain base-pairing (Figure 1).
Each tRNA serves as an 'adaptor" molecule that carries its specific amino acidcovalently attached to its 3’ end-to the site of protein synthesis.
C. Messenger RNA :
 Messenger RNAs are RNA molecules that carry the "message" from the DNA






to the ribosomes to be translated into protein.
The "message" in mRNA is carried in groups of three nucleotides called codons.
Each codon specifies one amino acid in a protein according to the rules of the
genetic code.
Messenger RNA (mRNA) comprises only about 5 % of RNA in the cell.
It is the most heterogeneous type of RNA in size (500 to 6000 nucleotides) and
base sequence.
The mRNA carries genetic information from the nuclear DNA to the cytosol
where it is used as the template for protein synthesis.
Special structural characteristics of eukaryotic mRNA (but not prokaryotic)
include a long sequence of adenine nucleotides (a 'poly-A tail) on the 3’ -end of
the RNA chain plus a 'cap' on the 5’ -end consisting of a molecule of 7methylguanosine attached 'backward' (5'5') through a triphosphate linkage
(Figure 2).
2
Figure 1 : A. Characteristic
tRNA structure B. Folded
tRNA structure
Figure 2:Termination of transcription in
eukaryotes: addition of poly(A) tails.
TRANSCRIPTION:
RNA is synthesized as a complementary strand to one of the DNA strands as a
template strand by the action of the enzyme RNA polymerase that reads the
template strand in the 3’  5’ direction and synthesizes RNA in the 5’ 3’. It
utilizes the ribonucleoside triphosphate as a building units.
The other DNA strand is called coding strand. A given strand may serve as
template strand for genes and coding for the other (Figure 3).
A central feature of transcription is that it is highly selective. For example many
transcripts are made of some regions of the DNA. In another regions, few or no
transcripts are made. This selectivity is due, at least in part, to signals embedded
in the nucleotide sequence of DNA.These signals instruct the RNA polymerase
where to start, how often to start, and where to stop transcription.
Another important feature of transcription is that many RNA transcripts that
initially are faithful copies of one of the two DNA strands may undergo various
modifications, such as terminal additions, base modifications, trimming, and
3
internal segment removal, followed by splicing, which convert the inactive
primary transcript into a functional molecule.
The structure of RNA polymerase, the signals that control transcription and
the varieties of modification that RNA transcripts can differ among
organisms and particularly from prokaryotes to eukaryotes.
Figure 3 : Expression of genetic
information by transcription
RNA POLYMERASES:
They are RNA-dependant RNA polymerases, i.e. they add (U) in the newly
synthesized strand for (A) in the template strand.
A) Prokaryotic:
 RNA polymerase is a multisubunit enzyme that makes RNA using DNA as a
template and then recognizes the end of the DNA sequence to be transcribed
(the termination region)..
 It uses the nucleoside triphosphates, ATP, GTP, CTP, and UTP to make a
complementary RNA copy of the DNA template strand.
4
 The nucleoside bases adenine, guanine, cytosine and uracil pair with the bases
thymine, cytosine, guanine, and adenine, respectively, in DNA to make RNA.
 Like DNA polymerases, RNA polymerases catalyze polymerization of
nucleotides only in the 5' to 3' direction antiparallel to its DNA template
strand(Figure 4).
 Unlike DNA polymerases, RNA polymerases do not require a primer to initiate
synthesis.
 A transcription unit extends from the promoter to the termination region and the
product of the process of transcription by RNA polymerase is termed the
primary transcript.
Figure 4 : Antiparallel, complementary base
pairs between DNA and RNA
 Transcription by RNA polymerase involves a core enzyme and several auxiliary
proteins :
1. Core enzyme: Four of the enzyme's peptide subunits. 2. 1. and 1 ’. are
responsible for the 5'3' RNA polymerase activity and are referred to as the core
enzyme (Figure 5).
 This enzyme lacks specificity, that is, it cannot recognize the promoter region
on the DNA template .
2. Holoenzyme: The  subunit ("sigma factor") enables RNA polymerase to
recognize promoter regions on the DNA.
 subunit + core enzyme  holoenzyme.
5
3. Termination factor: Some regions on the DNA that signal the termination of
transcription are recognized by the RNA polymerase itself. Others are recognized
by specific termination factors ( the rho (p) factor of E. coIi.)
Figure 5 : Prokaryotic RNA
polymerase
B) Nuclear RNA polymerases of eukaryotic cells
- There are 3 distinct classes of RNA polymerase in the nucleus of eukaryotic
cells. All are large enzymes with multiple subunits.
- Each class of RNA polymerase recognizes particular types of genes.
1. RNA polymerase I : This enzyme synthesizes the precursor of the large
ribosomal RNAs in the nucleolus. [ mRNA and tRNA are synthesized in the
nucleoplasm].
2. RNA polymerase II : This enzyme synthesizes the precursors of messenger RNAs
that are subsequently translated to produce proteins. Polymerase II also synthesizes
certain small nuclear RNAs (snRNA) and is used by some viruses to produce viral
RNA .
a. Promoters for class II genes: A sequence of DNA nucleotides that is almost
identical to that of the Pribnow box is usually found centered about 25
nucleotides upstream of the initial base of the transcription start site for an
mRNA molecule.
This consensus sequence is called the TATA or Hogness box (Figure 6).
Figure 6 :Eukaryotic gene promotor consensus sequences
6
b. Role of enhancers in eukaryotic gene regulation:
- Enhancers are special cis-acting DNA sequences that increase the rate of
initiation of transcription by RNA polymerase II.
- Enhancers must be on the same chromosome as the gene whose
transcription they stimulate .
- DNA sequences called "response elements" that bind specific transcription
factors called activators.
- By bending or looping the DNA, these enhancer-binding factors can
interact with transcription factors bound to a promoter and with RNA
polymerase II thereby stimulating transcription (Figure 7).
Silencers are similar to enhancers In that they act over long
distances to reduce the level of gene expression.
3. RNA polymerase III: This enzyme produces the small RNAs, including
tRNAs, the small 5S ribosomal RNA. and some snRNAs .
C) Mitochondrial RNA polymerase:
Mitochondria contain a single RNA polymerase that resembles bacterial RNA
polymerase more closely than it does the eukaryotic enzyme .
Figure 7: Some possible locations of
enhancers sequences
7
TRANSCRIPTION OF EUKARYOTIC GENES:
Transcription of eukaryotic genes is more complicated process than transcription in
prokaryotic cells, in addition to RNA polymerase recognizing the promoter region
and initiating RNA synthesis, several supplemental transcription factors bind to
distinct sites on the DNA either within the promoter region or some distance from it
(Figure 8).
Figure 8: The basic principle of Transcription
- Nuclear RNA polymerases of eukaryotic cells
- There are 3 distinct classes of RNA polymerase in the nucleus of eukaryotic
cells. All are large enzymes with multiple subunits.
- Each class of RNA polymerase recognizes particular types of genes.
POSTTRANSCRIPTIONAL MODIFICATION OF RNA :
- A primary transcript is a linear copy of a transcriptional unit -the segment of
DNA between specific initiation and termination sequences.
- The primary transcripts of both prokaryotic and eukaryotic tRNAs and rRNAs
are post-transcriptionally modified by cleavage of the original transcripts by
ribonucleases.
- tRNAs are then further modified to give each species its unique identity.
- In contrast. prokaryotic mRNA is generally identical to its primary transcript.
whereas eukaryotic mRNA is extensively modified posttranscriptionally .
A. Ribosomal RNA:
- Ribosomal RNAs of both prokaryotic and eukaryotic cells are synthesized from
long precursor molecules called preribosomal RNAs.
8
- The 23S. 16S. and 5S ribosomal RNAs of prokaryotes are produced from a
single RNA precursor molecule. as are the 28S. 18S. and 5..8S. rRNAs of
eukaryotes (Figure 9).
- The preribosomal RNAs are cleaved by ribonucleases to yield intermediatesized pieces of rRNA which are further "trimmed " to produce the required
ribosomal RNA species.
Figure 9: Posttranscriptional
processing of eukaryotic ribosomal
RNA by ribonucleases
B. Transfer RNA:
- Both eukaryotic and prokaryotic transfer RNAs are also made from longer
precursor molecules that must be modified.
- An intron must be removed from the anticodon loop and sequences at both the
5'- and the 3'-ends of the molecule must be trimmed.
C. Eukaryotic messenger RNA :
- The RNA molecule synthesized by RNA polymerase II (the primary transcript)
contains the sequences that are found in cytosolic mRNA.
- The collection of all the precursor molecules for mRNA is known
heterogeneous nuclear RNA (hnRNA).
- The primary transcripts are extensively modified in the nucleus after transcription.
- These modifications usually include :
1. 5’ “Capping":
- This process is the first of the processing reactions for hnRNA
- The addition of the guanosine triphosphate part of the cap is catalyzed
by the nuclear enzyme guanylyltransferase.
- Methylation of this terminal guanineoccurs in the cytosol, and is catalyzed by
guanine-7-methy!transferase.
9
2. Addition of a poly-A tail:
- Most eukaryote mRNAs have a chain of 40 to 200 adenine nucleotides
attached to the 3’-end (Figure 10) .
- This poly-A tail is not transcribed from the DNA.
- A consensus sequence called the polyadenylation signal sequence (AAUAAA)
found near the 3'-end of the RNA molecule signals that a poly-A tail is to be
added to the mRNA.
- These tails help stabilize the mRNAs and facilitate their exit from the nucleus.
Figure 10 : Posttranscriptional modification of mRNA showing
the cap and poly-A Tail
3. Removal of introns:
- Maturation of eukaryotic mRNA usually involves the removal of
RNA sequences which do not code for protein from the primary
transcript.
- The remaining coding sequences, the exons, are spliced together to form the
mature mRNA.
- The
a. molecular machine that accomplishes these tasks is known as the
spliceosome.
a. Role of small nuclear RNAs (snRNAs): snRNAs. in association
with proteins form small nuclear ribonucleoprotein particles
(snRNPs, or snurps"). These facilitate the splicing of exon
segments by forming base pairs with the consensus sequences at
each end of the intron.
b. Mechanism of excision of introns: The binding of snRNPs brings the
sequences of the neighboring exons into the correct alignment for
splicing.
c. Effect of splice site mutations: Mutations at splice sites can lead to
improper splicing and the production of aberrant proteins. It is estimated
that 15% of all genetic diseases are a result of mutations that affect
RNA splicing.
10
4. Alternative splicing of mRNA molecules:
- The pre-mRNA molecules from some genes can be spliced in two
or more alternative ways in different tissues.
- This produces multiple variations of the mRNA, and. Therefore, of its
protein product . This appears to be a mechanism for producing a diverse set
of proteins from a limited set of genes.
COMPLEMENTARY DNA (cDNA):
 The mRNA can be used as a template to make a complementary double




stranded DNA (cDNA) molecule using the enzyme reverse transcriptase.
Reverse transcriptase is an RNA-dependant DNA polymerase, i.e. it adds
(T) in the newly synthesized strand for (A) in the template strand.
Like all the other enzymes that synthesize nucleic acids, reverse
transcriptase moves along the template in the 3'5' direction,
synthesizing the cDNA product in the 5'3' direction but it lacks
proofreading activity.
The resulting cDNA is thus a double stranded molecule.
The cDNA can be amplified by cloning or by the polymerase chain
reaction (PCR).
RNA interference (RNAi) and (siRNA) :
RNA interference (RNAi) is a sequence-specific, posttranscription gene silencing
phenomenon that is triggered by double-stranded RNA and leads to the formation of
short(small) interference RNA molecules (siRNA). These siRNA molecules are genespecific and can be used in several clinical applications such as; gene therapy in
which downregulation of genetic activities in cancer cells and viruses leads to
interference with cancer cell proliferation and viral replication. Also, diagnosis of
normal and mutant gene function can be achieved through siRNA.
References:
 Lippincott’s Reviews of Biochemistry, 3rd edition by Champe PC,
Harvey RA, Ferrier DR, Lippincott William & Wilkins London, 2005
 Harper's Illustrated Biochemistry: 27th Edition by Murray RK, Granner
DK, Mayes PA, Rodwell VW, McGraw-Hill companies New York, 2005
 Text book of Biochemistry with Clinical Correlations 5th Edition, Devlin
TM Ed,Wiley –Liss New York 2002
 Essential Molecular Biology Review, Hall, P.W. , Blackwell Science,
Oxford.
 Achim Aigner. Delivery systems for the Direct Application of siRNAs to
induce RNA interference(RNAi) in Vivo, Journal of Biomedicine and
Biotechnology ; vol. 2006, Article ID 71659: 1-15
11