TRANSCRIPTION AND PROCESSING OF RNA

1. The steps of gene expression.

2. General characterization of transcription: steps, components of transcription apparatus.
3. Transcription of eukaryotic structural genes.
4. Processing of eukaryotic mRNAs.
5. Particularities of transcription of tRNA and rRNA genes in eukaryotes.
6. Transcription of mitochondrial genome.
7. Particularities of transcription in prokaryotes.

All of information about organisms is stored in DNA. Realization of information is made by

gene expression.
Gene expression represents the conversion of genetic information encoded in a gene into
RNA and protein, by transcription of a gene into RNA and (in the case of protein-coding genes) the
subsequent translation of mRNA to produce a protein. In both eukaryotes and prokaryotes there are
some steps of gene expression.
DNA replication The steps of gene expression in
DNA repair prokaryotic cells:
Genetic recombination - Activation and transcription of
genes
DNA
- Translation of mRNA – synthesis
of proteins
- Conformation – post-translational
modification of proteins.
DNA transcription
RNA synthesis The steps of gene expression in
eukaryotic cells:
RNA - Activation and transcription of
genes
- Processing of RNAs
- Export of RNAs from nucleus to
Codons Protein synthesis cytoplasm
- Translation of mRNA – synthesis
Protein of proteins
- Conformation – post-translational
modification of proteins.
Amino acids
Transcription is the first step in gene expression. Transcription represents the process of
complimentary synthesis of RNA from a DNA template.
Stretch of DNA that is transcribed as a single continuous RNA strand, a transcript, is called a
transcription unit. A unit of transcription may contain one or more sequences encoding polypeptides
(translational open reading frames (ORF) or cistrons). In prokaryotes polycistronic mRNAs are
common. In eukaryotes, monocistronic mRNAs are the general rule, but some transcription units
encode more than one polypeptide as a consequence of alternative transcriptional start sites and/or
alternative pathways of RNA splicing or other types of post- transcriptional RNA processing.
Components required for transcription:
- DNA molecule containing regulatory (promoter, terminator) and coding sequences
- RNA-polymerazes
- Specific transcription factors
- General transcription factors
- NTP (ATP, GTP, CTP, UTP).
Transcription is the principal point at which gene expression is controlled in both prokaryotes
and eukaryotes. There are three steps in the process of transcription:
 Transcription and processing
- Initiation (the most important)
- Elongation
- Termination

RNA-polymerases
RNA-polymerases are enzymes which catalyze the synthesis of RNA using DNA as template.
Direction of synthesis of RNA is 5'→3' and DNA is reading in direction 3'→5'. The strand of DNA
which serves as template is named sense chain, and the other strand, identical with RNA, is named
coding strand (Fig. 1).

Fig. 1. Transcription of RNA

The prokaryotic cells have only one type of RNA-polymerase, which synthesis all kinds of
RNAs.
The eukaryotic cells have three distinct classes of RNA-polymerases:
- RNA-polymerase I – the most active polymerase (50-70% of all cellular RNAs). It works
in nucleolus and synthesis rRNA 5,8S, 18S, 28S.
- RNA-polymerase II – works in nucleoplasme, synthesis mRNAs and snRNAs (10-40%).
- RNA-polymerase III – works in nucleoplasme, synthesis tRNAs and rRNA 5S (10%).

Transcription factors
There are molecules (usually proteins) that mediate transcription by interaction with DNA or
other proteins implicated in transcription. There are two types of transcription factors:
General transcription factors – are the same for all cell. Their functions:
- Facilitate the interaction between promoter and RNA-polymerase
- Participate in choosing of template strand and indicate direction of transcription
- Unwind and rewind double helix of DNA
- Prevent premature removing of RNA-polymerase from template
- Assure termination of transcription.
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 Transcription and processing
Specific transcription factors – are specific for each kind
of cells. They participate in decondensation of chromatin and bind specific to promoter, indicating

Transcribed region

sequence
Leading
Coding region Terminator
Promoter

Site of initiation Site of termination

of translation of translation
Point of initiation Site of
of transcription polyadenilation

Fig. 2. Structure of gene coding mRNA in eukaryotes

the active one.

Particularities of transcription of mRNA in eukaryotic cell

Initiation

Fig. 2 represents the structure

of gene coding for mRNA. For
initiation of transcription there are
some events, which consist in
activation of gene and beginning of
transcription:
- Decondesation of chromatin.
Demethylation of DNA.
- Interaction of specific factor of
transcription with promoter.
- Binding of TFIID (TBP) to
TATA-box (TF – Transcription
Factor, II – polymerase II; TBP –
TATA Binding Protein);
- TFIIA binds upstream from the
TBP. It stabilizes the complex
TBP-TATA-box.;
- In front of TBP binds TFIIB,
which unwind DNA using ATP;
- RNA-polymerase II, activated by
TFIIF binds to promoter. TFIIF is
a helicase, which unwind locally
DNA;
- After binding of factors TFIIE
and TFIIH RNA-polymerase can
move along the template strand of
DNA;
Fig. 3. Initiation of transcription
- Reading of first nucleotide from
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 Transcription and processing
template (+1) end incorporation of first ribonucleotide,
usually ATP;
- Initiation is finished by formation of first phosphodiester bond in newly synthesized RNA (Fig.
3).
Some distant sequences can also participate in the process of transcription. They may
facilitate the recognition of promoter by RNA-polymerase (enhancer) or interfere in this process
(silencer) (Fig. 4)
Regulatory protein Regulatory protein
attached to enhancer attached to promoter

Enhancer Promoter

Enhancer

RNA
polymeraze
II

Promoter
Fig. 4. Interaction of enhancer (via regulatory proteins) with promoter and RNA-polymerase

Elongation
- From RNA-polymerase are released TFIIB and TFIIE. TFIIF and FTIIH remain attached
to enzyme.
- RNA polymerase II reads DNA in direction 3'→5' and polymerizes RNA in direction
5'→3' (30 bases/sec)
- TFIIS prevents premature removing of RNA-polymerase
- At the promoter remain attached: FTIID, FTIIA that can interact with other RNA-
polymerase.

Termination
In Fig. 5 is shown
the structure of
terminator. It contains a
palindromic sequence a
region in which the
sequence on both strands
is identical when read in
an antiparallel direction.
After RNA-polymerase
transcripts the sequence
corresponding to the
terminator RNA forms a
Fig. 5. Structure of terminator
hairpin loop. RNA-
polymerase stops and a factor of termination rho (ρ) interact with enzyme and dissociate the complex
DNA-enzyme-RNA (Fig. 6).

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 Transcription and processing

RNA-polymerase
transcribes DNA

rho attaches to
recognition site on RNA

rho moves along RNA,

following RNA-
polymerase

RNA-polymerase pauses
at terminator and rho
catches up; rho unwinds
DNA-RNA hybrid

Termination:
RNA-polymerase, rho
and RNA are released

Fig. 6. Termination of transcription

Processing of RNA

The primary transcript represents an immature RNA. Processing of RNA represents the
events when the ends of RNA are modified and the non-coding sequences are removed from the pre
RNA (Fig. 7).

Fig. 7. The steps of processing of RNA

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 Transcription and processing
CAPing
The 5'-end of primary transcript is modified
during transcription. After 30 bases were
synthesized, guanilat-trasferase adds a methylated
GTP by unusual bond 5'-5' to the first nucleotide of
RNA (usually an Adenine). This structure
(7MeG5ppp5N) is named “CAP”. Also can be
methylated the next riboses in position 2' (Fig. 8).
CAP has the next functions:
 Stabilizes RNA due to unusual bond 5' -
5';
 Represents a site of recognition for
ribosome during initiation of translation.

Polyadenylation
After transcription the 3'-end of RNA contain
a palindromic loop, which is removed by excision in
the site AAUAAA. The enzyme poly(A)-
Fig. 8. The structure of CAP polymerase adds 100-200 residues of adenilic acid.
mRNA which cods for histones are not polyadenylated.
Poly(A)-tail has some functions:
 Assures the stability of 3'-end of RNA. Molecules that contain more long tails are more
stabile.
 Participates in passing of mRNA thru nuclear envelope.

Splicing
Splicing represents the process of removing of introns from pre mRNA and sealing of exons.
This process takes place with participation of
an enzymatic complex – splicesome.
Enzymes (U1-U6) represent
ribonucleoproteins and contain snRNA.
Introns are recognized by sequences GU at
5'-end and AG at 3'-end. There are some
steps in the process of splicing (Fig. 9):
 Site GU is recognized by U1;
 U2 binds to an Adenine from the interior
of intron (branch site);
 U4,U5,U6 associate to U1 and U2 forming
a loop by binding of 5'-end of intron to
Adenine via an unusual bond 5'–2';
 After removing of U4 3'-end of intron is
cleaved. The intron forms a lasso and is
removed together with proteins U2, U5,
U6;
Fig. 9. Splicing pre-mRNAs and assembly of spliceosomes
 The 3' and 5'-end of exons are sealed.
Resulting mRNA is transported to the cytoplasm, where will be used as template for protein
synthesis.
There are some types of splicing:
 Constitutive splicing – all introns are removed from pre mRNA and exons are sealed in the same
consecution as in gene.
 Alternative splicing – in mRNA remain only some of exons, and only some of introns are
removed. From one gene can be synthesized more types of proteins.
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 Transcription and processing

 Exon shuffling – change of consecution of exons in mRNA from the position in gene.
 Trans-splicing – exons from different pre mRNA participate to make one molecule of mRNA.

RNA editing
RNA editing is defined as a process responsible for any differences between the final
sequence of a messenger RNA (mRNA) and its genetically determined template. This process takes
place in cytoplasm and involves adding, removing or conversion of some nucleotides.

Particularities of I-st and II-nd class genes transcription

Eukaryotic ribosomes contain four RNA
molecules: 5S, 5,8S, 18S and 28S. In the
nucleolus there are several hundred copies
of transcription units which encod for 5,8S,
18S and 28S. The mammalian primary
transcript of I-st class genes is a 45S RNA
containing the sequences of 5,8S, 18S and
28S rRNAs. During maturation, the primary
transcript is cleft in 5 places, spacers are
Fig 10. Transcription and processing of rRNA removed and 3 types of rRNA are made (Fig
10).
The genes for the 5S rRNA is contained in a separate transcription unit. These genes are also
arranged in tandem: there are several repeating units
separated by untranscribed segments (Fig. 11). This
type of rRNA is transcribed by RNA-polymerase III.
Note that in higher eukaryotes the rRNA sequences
do not contain introns. Fig. 11. Organization of genes for the 5S rRNA
Eukaryotic tRNA molecules are also excised from large transcripts (called pre-tRNA), which
may contain one or more tRNA sequences. During processing the introns and spacer sequences are
removed, at the 3’ end a specific CCA sequence is added.

Transcription in mitochondria
In mammalian mitochondrial genomes are very compact; they are no introns. The 13 mRNA,
2 rRNA and 22 tRNA genes are under control of two promoters: HSP and LSP. Most genes are
expressed in the same direction and tRNA genes lie between the genes coding for rRNA or protein.
12 mRNA-coding, 2 rRNA-coding and 14 tRNA-coding regions are transcribed in clockwise
direction; 1 mRNA-coding and 8 tRNA-coding regions are read counter clockwise. Beginning with
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 Transcription and processing
the promoter DNA is transcribed into a single transcript, from
which RNAs are cleaved to release the mRNAs, rRNAs and tRNAs.

Transcription in prokaryotes
In bacteria and other prokaryotes, several genes may be grouped together to form a single
transcription unit under the control of a promoter – an operon. In an operon, genes encoding, for
example, the different enzymes of a metabolic pathway or the subunits of an enzyme complex, are
clustered and are transcribed together into a polycistronic transcript, under the control of a single
promoter. This transcript is then translated to give the individual proteins. Operons enable the rapid
and efficient coordinate expression of a set of genes required to respond to a change in the external
or internal environment (See Fig. 6 THE GENE).