Introduction

RNA synthesis (transcription) begins by uncoiling a section of DNA that will be

used as the template. RNA polymerase binds to a promoter sequence and initiates
separation of the DNA double helix. Complementary ribonucleotides align and
RNA polymerase catalyzes their polymerization. The newly synthesized RNA
strand undergoes post-transcriptional processing before it leaves the nucleus.

Ribonucleic acid (RNA)

Ribonucleic acid (RNA) is a polymeric molecule essential in various biological

roles in coding, decoding, regulation and expression of genes. RNA
and DNA are nucleic acids, and, along with lipids, proteins and carbohydrates,
constitute the four major macromolecules essential for all known forms of life.
Like DNA, RNA is assembled as a chain of nucleotides, but unlike DNA, RNA is
found in nature as a single strand folded onto itself, rather than a paired double
strand. Cellular organisms use messenger RNA (mRNA) to convey genetic
information (using the nitrogenous bases of guanine, uracil, adenine, and cytosine,
denoted by the letters G, U, A, and C) that directs synthesis of specific proteins.
Many viruses encode their genetic information using an RNA genome.

Structure of RNA

Each nucleotide in RNA contains a ribose sugar, with carbons numbered 1' through

5'.A base is attached to the 1' position, in general,  adenine (A),
cytosine (C), guanine  (G), or uracil (U).
 Chemical structure of RNA

Synthesis of RNA

Synthesis of RNA is usually catalyzed by an enzyme—RNA polymerase—using

DNA as a template, a process known as transcription. Initiation of transcription
begins with the binding of the enzyme to a promoter sequence in the DNA (usually
found "upstream" of a gene). The DNA double helix is unwound by
the helicase activity of the enzyme. The enzyme then progresses along the template
strand in the 3’ to 5’ direction, synthesizing a complementary RNA molecule with
elongation occurring in the 5’ to 3’ direction. The DNA sequence also dictates
where termination of RNA synthesis will occur.

Primary transcript RNAs are often modified by enzymes after transcription. For

example, a poly(A) tail and a 5' cap are added to eukaryotic pre-
mRNA and introns are removed by the spliceosome.
There are also a number of RNA-dependent RNA polymerases that use RNA as
their template for synthesis of a new strand of RNA. For instance, a number of
RNA viruses (such as poliovirus) use this type of enzyme to replicate their genetic
material. Also, RNA-dependent RNA polymerase is part of the RNA
interference pathway in many organisms.

An Overview of RNA Synthesis

RNA synthesis, or transcription, is the process of transcribing DNA nucleotide

sequence information into RNA sequence information. RNA synthesis is catalyzed
by a large enzyme called RNA polymerase. The basic biochemistry of RNA
synthesis is common to prokaryotes and eukaryotes, although its regulation is more
complex in eukaryotes. The close connection between prokaryotic and eukaryotic
transcription has been beautifully illustrated by the recently determined three-
dimensional structures of representative RNA polymerases from prokaryotes and
eukaryotes (Figure 28.1). Despite substantial differences in size and number of
polypeptide subunits, the overall structures of these enzymes are quite similar,
revealing a common evolutionary origin.
RNA Polymerase Structures.   The three-dimensional structures of RNA

polymerases from a prokaryote (Thermus aquaticus) and a

eukaryote (Saccharoromyces cerevisiae). The two largest subunits for each
structure are shown in dark red and dark blue. The similarity (more...)

RNA synthesis, like nearly all biological polymerization reactions, takes place in

three stages: initiation, elongation, and termination. RNA polymerase performs
multiple functions in this process:

1. It searches DNA for initiation sites, also called promoter sites or

simply promoters. For instance, E. coli DNA has about 2000 promoter
sites in its 4.8 × 106 bp genome. Because these sequences are on
the same molecule of DNA as the genes being transcribed, they are
called cis-acting elements.
2. It unwinds a short stretch of double-helical DNA to produce a single-
stranded DNA template from which it takes instructions.
3. It selects the correct ribonucleoside triphosphate and catalyzes the
formation of a phosphodiester bond. This process is repeated many times
as the enzyme moves unidirectionally along
the DNA template. RNA polymerase is completely processive—a
transcript is synthesized from start to end by a single RNA polymerase
molecule.
4. It detects termination signals that specify where a transcript ends.
5. It interacts with activator and repressor proteins that modulate the rate of
transcription initiation over a wide dynamic range. These proteins, which
play a more prominent role in eukaryotes than in prokaryotes, are
 called transcrip-tion factors or trans-acting elements. Gene expression is
controlled mainly at the level of transcription

The fundamental reaction of RNA synthesis is the formation of a phosphodiester

bond. The 3′-hydroxyl group of the last nucleotide in the chain nucleophilically
attacks the α-phosphate group of the incoming nucleoside triphosphate with the
concomitant release of a pyrophosphate (see Figure 5.25). This reaction is
thermodynamically favorable, and the subsequent degradation of the
pyrophosphate to orthophosphate locks the reaction in the direction of RNA
synthesis.

The chemistry of RNA synthesis is identical for all forms of RNA, including

messenger RNA, transfer RNA, and ribosomal RNA. The basic steps just outlined
also apply to all forms. Their synthetic processes differ mainly in regulation,
posttranscriptional processing, and the specific polymerase that participates.

RNA synthesis is a key step in the expression of genetic information. For

eukaryotic cells, the initial RNA transcript (the mRNA precursor) is often spliced,
removing introns that do not encode protein sequences. Often, the same pre-mRNA
is spliced differently. For eukaryotic cells, the initial RNA transcript
(the mRNA precursor) is often spliced, removing introns that do not encode protein
sequences. Often, the same pre-mRNA is spliced differently in different cell types
or at different developmental stages. In the image at the left, proteins associated
with RNA splicing (stained with a fluorescent antibody) highlight regions of the
newt genome that are being actively transcribed. [(Left) courtesy of Mark B. Roth
and Joseph G. Gall.)
The process of synthesizing RNA from the genetic information encoded by DNA
is called transcription. The enzymes involved in transcription are called RNA
polymerases. Prokaryotes have one type; eukaryotes have three types of nuclear
RNA polymerases.

The prokaryotic RNA polymerase consists of a core enzyme and an auxiliary

protein factor called sigma (s factor). The core consists of four subunits, two are
identical, a, the other two similar, b and b'. The b' subunit binds the DNA while
the b subunit binds the nucleotides that are to be joined together to form the RNA
molecule. Sigma factors function in identifying specific DNA sequences known as
promoters. Promoters are sites that tell the RNA polymerase where to begin
transcription.

Eukaryotes have four different RNA polymerases (RNA pol). Three are required
for transcription of nuclear genes and the fourth for transcription of mitochondrial
genes. RNA polymerase I transcribes ribosomal RNA (rRNA), pol II transcribes
mRNA and pol III tRNA and several small RNA's. The three polymerases consist
of ten or more subunits. All have two large subunits with homology to the b and b'
subunits of the prokaryotic RNA polymerase. The three eukaryotic polymerases
can be distinguished based on their sensitivity to a-amanitin, a toxin found in some
types of mushrooms. RNA pol II activity is severely inhibited, pol III weakly and
pol I is insensitive. The antibiotic rifampicin inhibits prokaryotic RNA
polymerases.
There are three phases of transcription: initiation, elongation and termination. It is
easier to understand the process by first examining elongation then initiation and
termination.

Elongation

RNA polymerase links ribonucleotides together in a 5' to 3' direction. The

polymerase induces the 3' hydroxyl group of the nucleotide at the 3' end of the
growing RNA chain which attacks (nucleophilic) the a phosphorous of the
incoming ribonucleotide. A diphosphate is released and the 5' carbon of the
incoming nucleotide is linked through a phosphodiester bond to the 3' carbon of the
preceding nucleotide.
Nucleotide incorporation is determined by base pairing with the template strand of
the DNA. The template is the DNA strand, also called the sense strand, that is
copied by the RNA polymerase into a complementary strand of RNA called the
transcript. The DNA strand that is not copied is know as the antisense strand. Note
that while the RNA chain grows in a 5' to 3' direction the polymerase migrates
along the sense strand in a 3' to 5' direction. Thus the 5' to 3' ribonucleotide
sequence of the RNA transcript is identical to the 5' to 3' antisense DNA strand
with uracil in place of thymidine.
Initiation

The initiation of transcription is directed by DNA sequences called promoters

which tell the RNA polymerase where to begin transcription. The subunits that
enable RNA polymerases to recognize and bind promoters are called initiation
factors. The initiating nucleotide can be either a purine or pyrimidine. There are
numerous eukaryotic promoters with multiple promoter sequence elements. Some
of the elements specify where transcription is to be initiated, others determine the
frequency with which transcription is initiated at a specific gene. The initiation of
transcription in eukaryotes is complicated and involves numerous factors (proteins)
that must interact with the DNA and with one another to initiate transcription.

Promoters

1. Only one strand of the DNA that encodes a promoter, a regulatory sequence,
or a gene needs to be written.
2. The strand that is written is the one that is identical to the RNA transcript,
thus the antisense strand of the DNA is always selected for presentation.
3. The first base on the DNA where transcription actually starts is labeled +1.
4. Sequences that precede, are upstream of the first base of the transcript, are
labeled with negative numbers. Sequences that follow the first base of the
transcript, are downstream, are labeled with positive numbers.

RNA pol II promoters are quite diverse. This enables the cell to choose and
regulate the expression of the 50 to 100 thousand different genes encoded by its
DNA. There are some sequence elements that are conserved and found in most
RNA pol II promoters. There are three "boxes": TATA usually found 25 to 35 base
pairs upstream, the CAAT box and the GC box both located from 40 to 200 base
pairs upstream.

These three elements provide a basal level of transcription and are found in most
"housekeeping" genes. Housekeeping genes encode enzymes and proteins that all
cell types require for normal function and are usually expressed at steady state or
basal levels. Other sequence elements, which are continually being discovered,
serve as regulatory elements. Elements that enable a cell to specifically turn other
non-housekeeping genes on or off in response to environmental signals such as
hormones, growth factors, metals and toxins. The spacing and orientation of all of
the sequence elements are critical for proper functioning. There is a third type of
sequence element that can be located either upstream or downstream relative to the
initiation site which is called an enhancer or silencer. Enhancers or silencers affect
the rate and frequency of initiation of transcription.

RNA pol III promoters for tRNA are found downstream of the initiation point.
These promoters consist of two elements, the first of which is located 8 to 30 base
pairs downstream and is called Box A. The second element is 50 to 70 base pairs
downstream and is called Box B.

RNA pol I promoter consists of a 70 base pair long core element and an upstream
element that is about 100 base pairs long. The core spans a segment of DNA that
includes sequences that are both up and downstream of the initiation site.

Termination

Prokaryotes use two means for terminating transcription, factor-independent and

factor-dependent. Certain DNA sequences function as signals that tell the RNA
polymerase to terminate transcription. The DNA of a terminator sequence encoded
an inverted repeat and an adjacent stretch of uracils. Factor-dependent termination
involves a terminator sequence as well as a factor or protein called rho. The
mechanisms by which eukaryotes terminate transcription are poorly understood.
Most eukaryotic genes are transcribed for up to several thousand base pairs beyond
the actual end of the gene. The excess RNA is then cleaved from the transcript
when the RNA is processed into its mature form.

RNA Processing

Most transcripts must be processed before becoming fully functional. Most

eukaryotic RNA must be transported across the nuclear membrane where it is
processed then transported to the cytosol. Processing helps stabilize and protect the
RNA so it can function in the cytosol and also functions in regulating the
expression of certain genes.

Mature mRNA is formed by extensively modifying the primary transcript also

called heterogeneous nuclear RNA (hnRNA). The hnRNA must undergo three
major modifications before maturing into mRNA: capping, polyadenylation and
splicing.

Capping: all mRNA's are capped at their 5' ends with 7-methylguanylate.
Guanylyl transferase catalyzes the linking of 7'-methylguanylate to the mRNA
through a 5' to 5' triphosphate bridge. The capping positions the mRNA onto the
40S preinitiation complex and protects it from exonuclease activity.

Polyadenylation: is the addition of a chain of adenylate residues, known as a poly

A tail to the 3' terminus of mRNA. After the RNA is cut, an enzyme poly A
polymerase, catalyzes the polymerization of adenylates. The poly A tail slows the
exonucleolytic degradation of mRNA, once the tail is removed mRNA is quickly
degraded.

Splicing: is the removal of noncoding sequences, derived from the DNA template,
from the hnRNA to form a functional mRNA. The noncoding sequences are called
introns while the coding sequences are known as exons. All introns have the
sequence GU at their 5' ends and AG at their 3' ends. The guanyl residue at the 5'
end of the intron is linked by a 2' to 5' phosphodiester linkage to an adenylate
residue within the intron. The result is a lariat (loop) structure and the release of
the 3' end of the first exon. The 3' end of the intron is spliced by an enzyme known
as a spliceosome, which releases the loop and frees the 5' end of the second exon.
The exons are then joined together.

The rRNA of both prokaryotes and eukaryotes are synthesized as large precursors.

The precursor rRNA's are processed into their mature form by nucleases and
methylases.

The tRNA's of both prokaryotes and eukaryotes are also transcribed as precursors

which are cleaved and extensively modified.

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