Post-translational modification
RNA structure
Ribosomes - Protein Synthesis
Protein synthesis gives a more basic summary and overview of protein synthesis and makes a good starting point for those who are
new to this topic. The article below is more detailed.

See also: the cell nucleus and DNA

Ribosomes are factories that manufacture proteins for cells. They are small (diameter about 10 nm or 10 millionths of a millimetre)
ribonucleoprotein enzymes. A ribonucleoprotein is a structure consisting of protein and RNA (ribonucleic acid).

In prokaryotes, the cytoplasm surrounding the nucleoid is rich in risosomes (and is called the riboplasm) - in bacteria proteins that
make up the ribosome are the most abundant proteins in the cytosol.

In eukaryotes ribosomes can exist free in the cytosol or bound to endoplasmic reticulum (forming rough endoplasmic reticulum, or
RER, so-called because the ribosomes stud its outer/cytosolic surface). The cytosol has a high gluathione concentration, which
makes it a reducing environment, and so proteins with disulphide bridges (-S-S-) can not be made here (since the disulphide bridge
would remain reduced to hydrogen sulphide groups (-SH + HS-). However, proteins containing one or more disulphide bridges can
be manufactured in the RER. Proteins destined for the plasma membrane are also manufactured in the RER, so that they can be
readily packaged into membrane-bound vesicles and enter the membrane-transport system.

Ribosomes are comprised of two structural subunits: the large subunit and the small subunit. These subunits are designated by
their sedimentation values, that is the time they take to sink through solution, as in a centrifuge, in units called
Svedbergs (S).
Note that although related to size, Sveddbergs measure sedimentation time and so will depend on density and probably
interactions with water molecules.

  • In eukaryotes the ribosome, an 80S ribosome, is made of a 40S small subunit + a 60S large subunit.
  • In prokaryotes the ribosome is 70S and made of a 50S large subunit + a 30S small subunit.
  • Eukaryotic chloroplasts and mitochondria contain prokaryote-like 70S ribosomes.

The composition of these subunits, in terms of RNA molecules (as measured in Svedbergs, S, and number of nucleotides, nuc) and
proteins can be summarised as follows:

50S: 5S RNA (120 nuc), 23S RNA (2900 nuc), 34 proteins
30S: 16s RNA (1540 nuc), 21 proteins

60S: 5S RNA (120 nuc), 28S RNA (4700 nuc), 5.8 RNA (160 nuc), about 49 proteins
40S: 18S RNA (1900 nuc), about 33 proteins

The ribosomal RNAs are often designated rRNA. As you can see, ribosomes are complex structures (ribonucleoprotein enzyme
complexes). Enzymes are biological catalysts which speed up the rate of chemical reactions, each enzyme being specifically
responsible for one reaction (or a few related reactions). Enzymes lower the activation energy of a reaction, so that it occurs faster
at lower temperatures, and so reduce the time taken for a reaction to reach equilibrium. Most enzymes are proteins, but RNA
molecules can be enzymes too.

The ribosome is not only complex, but it is also the most important part of the biological cell, along with the necessary information
needed to make proteins (encoded in DNA or RNA). Ribosomes make proteins and proteins make cells. Ribosomes may well
predate the role of DNA on an evolutionary timescale.

Protein Synthesis

Involves three stages:

1. Transcription
2. Post-transcriptional modification
3. Translation

1. Transcription

The instructions to make the required proteins are encoded in the DNA in the cell nucleus (or nucleoid of prokaryotes). The first
process is transcription, in which this information is copied (transcribed) onto a molecule of RNA, called messenger RNA (mRNA).
The double helix of the DNA is broken open (by the enzymes
DNA helicase, which breaks the hydrogen-bonds between the
strands, and
DNA topoisomerase, which unwinds the DNA to prevent tangles) at the gene to be transcribed, forming an open
transcription bubble. RNA polymerase reads the coding strand of the DNA and synthesises RNA that is complimentary to the
DNA coding strand.

RNA polymerase reads the DNA in the 3' to 5' direction and so synthesises RNA in the complimentary 5' to 3' direction.
Above: the structure of RNA. Green circles: phosphate groups; orange pentagons: sugar groups (ribose); rectangles: bases. The
5' end is uppermost, the 3' end lowermost. Compare to the structure of DNA.

  • RNA is a polynucleotide, like DNA. However, whereas DNA is usually found in the double-stranded form (with the two
    strands entwined together in a double-helix) RNA is usually single-stranded.

  • Both a single-strand of DNA and RNA have a phosphate-sugar backbone, in RNA the sugar is ribose (a 5-carbon, 5C,
    sugar) whereas in DNA the sugar is deoxyribose (ribose minus one of its oxygen atoms, i.e. deoxygenated ribose).

  • The bases in DNA are adenine (A), guanine (G), thymine (T) and cytosine(C).
  • In RNA, the base uracil (U) replaces thymine.

  • Like DNA, each strand of RNA has a 5' (5-prime) end where it terminates in a phosphate group (-PO3) and a 3' (3-prime)
    end where it terminates in a hydroxyl (-OH group).

Although the primary structure of RNA is a single-strands polynucleotide (a polymer of nucleotides) parts of the molecule can pair
with itself, by forming hydrogen bonds between complimentary base-pairs, in a way similar to the pairing between the two strands
of DNA. This 'intramolecular base-pairing' gives rise to secondary structures, such as the
stem-loop (hairpin loop):
A pseudoknot is another secondary RNA structure, consisting of 2 or more stem-loops with half of one stem intercalated
between the two halves of the other stem-loop. For example, this structure is adopted by the genome of the turnip yellow
mosaic virus.

Polynucleotides also have tertiary structural motifs, that is 3D structures formed from one or more secondary structural motifs.
(A motif is a pattern). This is important to the function of many RNA molecules, including RNA enzymes (
ribozymes) for

  • Double helix (e.g. DNA)
  • Major and minor groove triplexes - formed by the insertion of nucleotides into the major and minor grooves of the double
  • Quadriplexes - e.g. quadruple helix in DNA and RNA
  • Coaxial stacking in RNA, in which two double-helixes are arranged in series with their bases stacked one-upon-the-other
    at the junction between the helices. This occurs in many ribozymes, self-splicing introns and in the tRNA for

Non-coding functional RNAs: mRNA is a coding RNA (it is translated into polypeptide). Other RNAs are non-coding and are
not translated, but still have important functions, e.g. rRNA, tRNA and
microRNA. The microRNAs are short (about 20
nucleotides long) and regulate the translation of mRNAs by the ribosome. The transcripts of about 30% of protein-coding
genes in mammals are thought to have their transcrtipts regulating by microRNAs. The  Piwi-interacting RNAs, which associate
with proteins called piwi proteins which help regulate splicing, especially in spermatogenesis (sperm cell production).

Transfer RNA (tRNA)

Transfer RNA consists of 4 stem-loop structure and a single-stranded region which can be ester bonded to an amino acid (by
an aminoacyl-tRNA synthetase enzyme). [An ester bond is an oxygen atom bridge between two carbon atoms in which one of
the carbon atoms is a C=O or carbonyl group:  -C-O-C=O.] Each tRNA carries a specific sequence of three nucleotides, called
an anticodon. This sequence matches its complimentary codon (triplet of nucleotides) representing an amino acid in the
2. Post-transcriptional modification

The RNA synthesised by the RNA polymerase (specifically known as the DNA-dependent RNA polymerase) is called the primary
. This pre-mRNA must leave the nucleus and carry its instructions to the ribosome. The most important of these
modifications in eukaryotes is
splicing. In eukaryotes, a gene typically contains polypeptide coding regions, called exons,
instrespersed with non-coding regions called
introns. The introns are removed in an enzymic reaction called splicing and the
exaons joined or spliced together in order to produce a continuous, uninterrupted polypeptide code.
Messenger RNA (mRNA)

Usually the codon AUG signifies a start codon, in both prokaryotes and eukaryotes, where translation by the ribosome should

In prokaryotes, there is a sequence of nucleotides on the mRNA, called the
Shine-Dalgarno sequence (SDS) (or
Shine-Dalgarno box) which always consists of the consensus sequence AGGAGG followed by a variable sequence. (In
Escherichia coli, the complete sequence is AGGAGGU). This sequence is located 8 b (b = bases or nucleotides) upstream
(i.e. not downstream where the message to be read is located) of the start codon. This sequence aligns the ribosome with the
AUG start codon and binds the 16S RNA of the small 30S subunit, to a sequence on the 16S rRNA called the
anti-Shine-Dalgarno sequence (ASDS). When the SDS binds to the ASDS, the three protein initiation factors (IF1, IF2 and IF3)
bind the soS subunit and the initiator tRNA coding for fMet binds the ribosome. Note that in prokaryotes, the first amino acid of
a polypeptide is not methionine but a modified form of methionine, called
formyl-methionine (f-methionine) - methionine with
a formyl group bound to it. [A formyl group is -CHO, the aldehyde group). There is an additional
ribosome-binding site
15-30 nucleotides upstream of the start codon.

In eukaryotes, the equivalent is a different short sequence of nucleotides called the
Kozak sequence, located 3 b upstream
of the AUG start codon. Additionally, the 5' cap of the mRNA forms the ribosome binding site.

The tables below summarise the amino acids (and their abbreviations) coded for in the
genetic code and the codon
sequences on the mRNA that code for each.
Above a stem-loop.
In RNA: A always pairs with U and G with C.
Introns may also occur in prokaryotes, though they are rare. Some introns form RNA enzymes (ribozymes) which catalyse their own
removal or splicing from the transcript - so-called
self-splicing introns. Others encode proteins/enzymes that facilitate intron
splicing. Some are apparently
selfish genetic elements and encode an endonuclease which can cut them out and move them to
other nucleotides, as
mobile genetic elements. A spliceosome is an enzyme complex of RNA and protein molecules which
splices out introns. Spliceosomes occur in both the nucleus and cytosol. Some introns also contain
regulatory elements that
control transcription, for example

Promoter: a sequence of DNA upstream from the coding sequence of a gene (or group of genes) that acts as an off-on switch for
transcription. RNA polymerase binds to the promoter which is upstream of the gene and on the same strand of DNA (said to be a
cis-regulatory element, meaning on the same strand).

Enhancer: another regulatory sequence of DNA that may occur inside an intron, enhancers are similar to promoters but help turn
transcription rates up or down by acting at a distance from the gene or genes they effect.

Single-stranded RNA and DNA molecules have direction, like any code they must be read in the right direction. One end of the
RNA molecule is called the 5' (5-prime) end and the other the 3' (3-prime) end. Complimentary nucleotide strands are aligned in
the opposite direction in double-stranded nucleotides.

Other post-translational modifications

Apart from splicing out introns, the pre-mRNA is modified in other ways.

5' capping

The 5' or phosphate-bearing end of mRNA is typically modified. In eukaryotes a guanine (G) nucleotide is added to the end in the
'wrong-way round' as it were, with its 5' end bonding to the 5' end of the RNA (creating a phosphate to phosphate linkage, actually
the link contains three phosphate groups in a row and is called a 5' to 5' triphosphate bridge) and then the guanine is methylated
(a methyl or -CH3 group is added to it) by an enzyme called methyl transferase. The result is a
7-methylguanylate cap which
looks like the 3' end of an RNA strand. This 5'-cap serves to protect the mRNA from enzymes in the cytosol which normally destroy
RNA from the 5' end. This is vital if the mRNA is to last long enough to be translated. (The 5' cap does have other functions, like
assisting transport of the mRNA across the
nuclear envelope through the nuclear pores). This capping occurs during transcription.

3' capping

The 3' end of the RNA in eukaryotes also needs protection from enzymatic degradation. This is achieved by the addition of about
200 adenine (A) residues, forming a
poly-A tail.


In prokaryotes there is rarely any post-transcriptional processing of the primary transcript, which functions as mature mRNA.
3. Translation

  • After post-transcriptional modifications are completed, the RNA transcript is now mature messenger RNA (mRNA). This
    mRNA is translated by ribosomes in the cytoplasm (either free in the cytosol or attached to rough endoplasmic reticulum

  • The two subunits of the ribosome, which are separate from one-another when not in use, assemble on the mRNA molecule
    at the correct end of the molecule (the start of the message).

  • The nucleotides/bases on the mRNA form a message, which is a code for the amino acid sequence of the polypeptide to be
    made. This code is grouped into 'words' called codons. Each codon is three nucleotides long and codes for one amino acid
    (of which there are 20 types).

  • As the code on the mRNA is translated, the correct amino acid is inserted into the growing polypeptide chain, with the help
    of transfer RNA (tRNA) molecules. Each tRNA contains a sequence of three unpaired bases called an anticodon, which
    matches the codon for a specific amino acid. Each tRNA molecule is bound to an amino acid by an enzyme called an
    aminoacyl tRNA synthetase. The correct amino acid is added to each tRNA to match its specific anticodon.

  • Initially, proteins called initiation factors (IFs) bind to the small ribosomal subunit and help it bind to the mRNA at the
    correct place and to help the assembled ribosome begin reading from the start codon (usually AUG, coding for the amino
    acid methionine).

  • The large ribosomal subunit has two sites - a P-site (peptidyl site) where an amino acid is added to the growing
    polypeptide chain by formation of a peptide bond and an A-site (aminoacyl site) where the next tRNA arrives, waiting for
    the P-site to become available. When the amino acid is added to the chain at the P-site, the empty tRNA is detached from
    the site by the ribosome complex and the ribosome moves one codon (three nucleotides) along the mRNA, to the next
    codon in the message.

  • The start codon is always near the 5' end of the mRNA:

  • Ribosomes always read the mRNA from its 5' end to its 3' end.

In the diagram below the 5' end of the mRNA is on the left and the ribosome is moving to the right.
The ribosome continues until it reaches a stop codon. At this point translation stops, the ribosome detaches from the mRNA and
disassembles and the polypetide chain is released (either to the cytosol or to the lumen of the RER). More ribosomes may already
be reading the mRNA molecule, lined-up in a queue called a
polysome. Eventually the mRNA molecule is destroyed.
The Structure of RNA
Example: Methionine (Met) is coded for by the codon AUG, on the mRNA, which also functions as a start codon if it occurs
in the right place near to the ribosome-binding sequence.

Referring to the tables and to the
structure of DNA the questions below (and their answers in blue) will hopefully make
sense to you:
Where in Q.2 we have defined the coding strand of the DNA molecule to be that strand which the RNA polymerase reads during
Enzyme Complexes

The scientific view of the cell has changed considerably over recent years, from something akin to a 'bag of chemicals' in which
biochemical reactions occurred in a biochemical soup to one in which the cell is seen as a very highly-ordered and compartmentalised
biochemical and biomechanical machine. Enzymes that work together to regulate the same set of chemical reactions group together
to form enzyme complexes (which may be transient) which are nano-machines with intricate and minute moving mechanical and
chemical parts.

One example is the
ribosome, along with the initiation factors. The abundance of ribosomes and their large size made this one of the
first enzyme complexes to be realised. Other examples are:

Replisome: involved in unzipping, unwinding and replicating DNA. Includes DNA polymerase (DNA-dependent DNA polymerase).

Spliceosome: for splicing pre-mRNA.

transcription complex that transcribes DNA into RNA. Includes RNA polymerase (DNA-dependent RNA polymerase).

Proteasome: found in the nucleus and cytosol of eukaryotes and some prokaryotes, these complex degrade damaged, unneeded
and worn-out proteins.

There are other examples and more are being identified all the time. A structure like a ribosome is very complex and yet very minute,
which makes a complete understanding of how it works difficult to achieve, however, rapid progress is being made, helped by
techniques like
cryo-electron microscopy, in which the rapid freezing of cells preserves their structures with high precision, allowing
a series of ultra-thin sections to be analysed, averaged on computer and reconstructed into a 3D image using computer software.