1. Transcription is the process by which DNA is copied into messenger RNA (mRNA) by RNA polymerase. This involves three phases - initiation, elongation, and termination. 2. Eukaryotic transcription is more complex than prokaryotic transcription due to multiple RNA polymerases, nucleosomes, separation of transcription and translation, and intron-exon structure of genes. 3. Following transcription, eukaryotic mRNA undergoes processing including capping, polyadenylation, and splicing before being translated into protein by ribosomes.

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Protein synthesis
o Protein synthesis : Transcription (DNA makes RNA) +Translation (RNA makes protein)
o Transcription (DNA makes RNA): is the process by which, the nucleotide sequence of
one strand of DNA is transcribed into a complementary molecule of RNA (messenger
RNA, mRNA). The DNA helix is opened by a complex set of proteins.
o The DNA strand in the 3' to 5' direction (noncoding, template, antisense or minus strand)
serves as the template for the transcription of DNA into RNA by RNA polymerase. RNA
is synthesized in the 5' to 3' direction.
o Transcription occurs in three phases – initiation, elongation and termination.
 Initiation: RNA polymerase binds to specific DNA sequences called promoters to
initiate RNA synthesis to the 3' side of the promoter. The RNA polymerase binds to
the dsDNA at a promoter sequence, resulting in local DNA unwinding. The position
of the first synthesized base of the RNA is called the start site and is designated as
position +1.
 Elongation: RNA polymerase moves along the DNA and sequentially synthesizes the
RNA chain. DNA is unwound ahead of the moving polymerase, and the helix is
reformed behind it.
 Termination: RNA polymerase recognizes the terminator which causes no further
ribonucleotides to be incorporated. Some terminators require an accessory factor
called rho for termination.
prokaryotic structural gene eukaryotic structural gene

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Transcription in Eukaryotes
 The process of activating gene expression in eukaryotes is far more complex than in
prokaryotes. There are several differences between eukaryotes and prokaryotes that
impinge upon gene activation and RNA processing.
• Eukaryotes possess multiple RNA polymerase enzymes.
• The wrapping of DNA into nucleosomes represents a barrier to transcription.
• The physical separation of the nucleus (where transcription occurs) and the cytoplasm
(where translation occurs) in eukaryotes means that mechanisms must exist to protect
the mRNAs from degradation in the cytoplasm before they have been translated.
• Eukaryotic genes are monocistronic (one gene one mRNA) while in prokaryotic
monocistronic and polycistronic (more than one gene one mRNA )
• The genes of eukaryotes are not continuous and are split into coding regions and non-
coding regions.
RNA Processing
(Capping, polyadenylation and splicing)
 Capping: the 5'end of the message is capped by the addition of a 7-methyl guanosine
residue. The function of the cap is not entirely clear. It has been suggested that the cap,
and proteins that bind to it, direct ribosome binding and correct translational initiation.
 Polyadenylation: the 3'end of the mRNA is cleaved from the extending chain and then
polyadenylated. The process of polyadenylation is carried out by an enzyme called polyA
polymerase (PAP).
 RNA Splicing: The capped and polyadenylated mRNA is spliced to remove all introns
(non-coding regions) and fuse all exons (coding regions) together into a single unit that
can be translated.
Spliceosomes is complexes contain both RNA and protein components, and are found
exclusively in the nucleus, responsible for splicing.
 Alternative Splicing
o The capped and polyadenylated mRNA is Alternative spliced to remove all introns with
number of exons and fuse remaining exons together into a single unit that can be
translated.(one gene coding for more than on protein)
o Alternative splicing therefore greatly increases the number of different protein activities
that can be generated from a defined set of genes within the genome.
RNA editing: The sequence of an mRNA molecule may be changed (Individual nucleotides
may be substituted, added or deleted) after synthesis and processing by RNA editing

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Translation (RNA makes protein)
 Ribosomes: complex of rRNAs and proteins that are sites of protein synthesis. The
overall structure and function of eukaryotic ribosomes is very similar to those from
bacteria. Each ribosome consists of two subunits, a small subunit and a large subunit. It
found on RER and nuclear envelope or as free in cytoplasm.
 tRNA : The tRNAs transport a specific amino acid to the mRNA triplet it encodes. The
tRNAs are small molecules, which form distinctive clover leaf secondary structures by
internal base pairing. The stem-loops of the clover leaf are known as arms:
 the anticodon arm contains in its loop the three nucleotides of the anticodon which will
form base pairs with the complementary codon in mRNA during translation;
 the D or DHU arm (with its D loop) contains dihydrouracil, an unusual pyrimidine;
 the T or T C arm (with its T loop) contains another unusual base, pseudouracil
(denoted ) in the sequence T C;
 Some tRNAs also have a variable arm (optional arm).
 The amino acid acceptor stem. This is where the amino acid becomes attached, at the
3’ OH group of the 3-CCA sequence.
Enzymes called aminoacyl-tRNA synthetases couple the amino acid encoded by a
particular codon to the tRNA that contains the appropriate anticodon. The codon–anticodon
pairing therefore directs the addition of the correct amino acid to the growing polypeptide
chain. Then RNA is translated upon ribosomes.

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 Genetic code
o Genetic code: is the rules that specify how the nucleotide sequence of an mRNA is
translated into the amino acid sequence of a polypeptide.
 The sequence of the mRNA is read in groups of three nucleotides called codons, with
each codon specifying a particular amino acid.
 Stop or Termination codons (UAG, UGA and UAA) do not encode an amino acid.
Whenever one of these codons is encountered by a ribosome, it leads to termination
of protein synthesis.
 Start or initiation codon :( AUG) codes for methionine. Although methionine is found
at internal positions in polypeptide chains, all eukaryotic polypeptides also start with
methionine and all prokaryotic polypeptides start with a modified methionine (N-formyl
methionine).
o The degeneracy of the genetic code: means that more than one codon will result in the
insertion of an individual amino acid into a growing polypeptide chain. Since RNA is
composed of four types of nucleotides, there are 43 = 64 possible codons, that is 64
possible triplets of nucleotides with different sequences. Most amino acids (20 different
amino acids) in proteins are specified by more than one codon
o Reading frames: The mRNA sequence can be read by the ribosome in three possible
reading frames. Usually only one reading frame codes for a functional protein since the
other two reading frames contain multiple termination codons. In some bacteriophage,
overlapping genes occur which use different reading frames.
o Open reading frame (ORF): is a run of codons that starts with ATG and ends with a
termination codon, TGA, TAA or TAG. Coding regions of genes contain relatively long
ORFs unlike noncoding DNA where ORFs are comparatively short. The presence of a
long open reading frame in a DNA sequence therefore may indicate the presence of a
coding region. Computer analysis of the ORF can be used to deduce the sequence of the
encoded protein.
Translation process
o Translation is making a protein using the information provided by messenger RNA.
o During translation, the sequence of an mRNA molecule is read from its 5’ end by
ribosomes which then synthesize an appropriate polypeptide. Both in prokaryotes and
in eukaryotes, the nucleotide sequence of the coding DNA strand, 5’ to 3’, specifies in
exactly the same order the amino acid sequence of the encoded polypeptide, N-
terminal to C-terminal.

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The mechanism of translation can be split into three steps – initiation, elongation and
termination.
 In bacteria, Initiation begins when the ribosome assembles on the purine-rich, Shine–
Dalgarno sequence. In eukaryotes, ribosome assembly occurs at the Kozak
sequence. The ribosome then moves along the mRNA in a 5' to 3' direction.
Translation starts at the AUG initiation codon and continues along the mRNA until the
ribosome dissociates when it reaches the termination codon. However, more than one
ribosome may be bound to single mRNA at one time, forming a polysome. The
number of ribosomes bound to a particular mRNA will influence the rate of its
translation: the higher the number of ribosomes bound, then the higher will be the
number of protein chains produced.
Posttranslational processes
Translation of an mRNA sequence into an amino acid sequence on the ribosome is not the
end of the process of forming a functional protein. To function,
 Protein folding and modification
 Correct folding into its three dimensional conformation
 Binding to any cofactors required
 Assemble with its partner protein chains (if any)
 Protein glycosylation
 Protein phosphorylation
 Removal of methionine residue at the amino-terminal end of the newly synthesized
polypeptide by methionine aminopeptidase .
 Protein targeting
In eukaryotes, all proteins are synthesized on ribosomes that are located in the cytoplasm.
However, some of these proteins end up in the nucleus, in the mitochondria, anchored in the
membrane, or secreted from the cell
 Soluble cytosolic proteins are simply released from the ribosome after polypeptide
synthesis is complete, and are already in the correct location to undertake their specific
function.
 Many eukaryotic proteins are, however, destined for a particular compartment of the cell.
The polypeptide itself encodes the information required for its final destination. Eukaryotic
proteins that are destined to accumulate within the nucleus contain a nuclear localization
signal (NLS), consisting of a short stretch of predominately basic amino acids (e.g.
PKKKRLV), which direct the protein into the nucleus.
 The proteins are synthesized with short sequences that target the protein to the correct
place or cellular compartment. For example, membrane proteins or proteins that are
secreted from the cell are synthesized with a short leader peptide, called a signal
sequence, at the amino-terminal end. This stretch of 15 to 25 amino acids is recognized
by membrane proteins that transport the new protein through the cell membrane; in this
process, the signal sequence is cleaved by a peptidase.

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Figure ( ) Signal sequences. Proteins destined to be secreted from the cell have an amino-
terminal sequence that is rich in hydrophobic residues. This signal sequence binds to the
membrane and draws the remainder of the protein through the lipid bilayer. The signal
sequence is cleaved from the protein in this process by an enzyme called signal peptidase.