3. • The flow of genetic information is unidirectional and requires two major
steps: transcription and translation.
• First, the information of the gene is transcribed into an intermediary RNA
molecule, which is synthesized in sequences that are precisely
complementary to those of the strand of DNA (transcription).
• During the second major step the sequence information in the messenger
RNA molecule (mRNA) is translated into a corresponding sequence of
amino acids (translation).
• The length and sequence of the amino acid chain specified by a gene
results in a polypeptide with a biological function (gene product) or The
proteins which do most of the work in the cell.
• The main concept in the central dogma is that DNA does not code for
protein directly but acts through an mediator molecule called ribonucleic
• Exceptions To The Central Dogma Exist: Reverse TranscriptionExceptions To The Central Dogma Exist: Reverse Transcription
• Some RNA viruses, called “retroviruses” make a DNA copy of themselves
using the enzyme reverse transcriptase.
• The DNA copy incorporates into one of the chromosomes and becomes a
permanent feature of the genome.
• The DNA copy inserted into the genome is called a “provirus”. This
represents a flow of information from RNA to DNA..
4. Transcription and translation in eukaryotic cells are separated in space andTranscription and translation in eukaryotic cells are separated in space and
Extensive processing of primary RNA transcripts in eukaryotic cells.Extensive processing of primary RNA transcripts in eukaryotic cells.
• Transcription is the synthesis of RNA molecules, with DNA as a template, and it is
the first step in the transfer of genetic information from genotype to phenotype. The
process is complex, and requires a number of protein components.
• The enzyme used in transcription is “RNA polymerase”.
• There are several forms of RNA polymerase.
• In eukaryotes, most genes are transcribed by RNA polymerase II.
• The raw materials for the new RNA are the 4 ribonucleoside triphosphates:
– ATP, CTP, GTP, and UTP.
• As with DNA replication, transcription proceeds 5’→ 3’: new bases are added to
the free 3’ OH group.
• Unlike replication, transcription does not need to build on a primer.
• Instead, transcription starts at a region of DNA called a “promoter”.
• For protein-coding genes, the promoter is located a few bases 5’ to (upstream from)
the first base that is transcribed into RNA.
• Promoter sequences are very similar to each other, but not identical. If many
promoters are compared, a “harmony sequence” can be derived. All promoters would
be similar to this harmony sequence, but not necessarily identical.
6. Classes of RNAClasses of RNA
• Ribosomal RNA (rRNA), along with ribosomal protein subunits, makes up the
ribosome, the site of protein assembly.
• Messenger RNA (mRNA) carries the coding instructions (specific sequence
=codons) for synthesizing polypeptide chains from DNA to the ribosome. Large
precursor molecules, which are termed pre-messenger RNAs (pre-mRNAs), are the
immediate products of transcription in eukaryotic cells. Pre-mRNAs are modified
extensively before they exit the nucleus for translation into protein. Prokaryotes do
not possess premRNA; in these cells, transcription takes place concurrently with
• Transfer RNA (tRNA) serves as the link between the coding sequence of
nucleotides in the mRNA and the amino acid sequence of a polypeptide chain.
• Each tRNA attaches to one particular type of amino acid and helps to incorporate
that amino acid into a polypeptide chain.
• Additional classes of RNA molecules are found in the nuclei of eukaryotic cells.
• Small nuclear RNAs (snRNAs) combine with small nuclear protein subunits to form
small nuclear ribonucleoproteins (snRNPs, affectionately known as “snurps”). The
snRNPs are analogous to ribosomes in structure, only smaller, and they typically
contain a single RNA molecule combined with approximately 10 small nuclear
protein subunits. Some snRNAs participate in the processing of RNA (spicing=
removal of introns)
• Small nucleolar RNAs (snoRNAs) take part in the processing of rRNA.
• Small cytoplasmic RNAs (scRNAs) Small RNA molecules that found in the
cytoplasm of eukaryotic cells are associated with rough endoplasmic reticulum and
involved in some protein functions.
8. Process of TranscriptionProcess of Transcription
• Transcription starts with RNA polymerase binding to the promoter.
• Various other proteins (transcription factors) help RNA polymerase bind to the
promoter. Other DNA sequences further upstream from the promoter are also
• Once it is bound to the promoter, RNA polymerase unwinds a small section of the
DNA and uses it as a template to synthesize a complementary RNA copy of the
• The DNA strand used that transcribed is called the template.
• the other strand (non-transcribed) is the “coding strand???!!!!”.
• Notice that the RNA is made from 5’ →3’ end, so the template strand is actually
read from 3’ → 5’.
• The mRNA transcribed from the template DNA is called the RNA sense strand.
• RNA transcribed under experimental conditions from the opposing DNA strand is
called antisense RNA.
• RNA polymerase proceeds down the DNA, synthesizing the RNA copy.
• In prokaryotes, each RNA ends at a specific terminator sequence.
• In eukaryotes transcription doesn’t have a definite end point; the RNA is given a
definitive termination point during RNA processing.
10. •The nucleotide in the template strand at which transcription begins is designated
with the number +1.
• Transcription proceeds in the downstream direction, and nucleotides in the
transcribed DNA are given successive positive numbers.
•Downstream sequences are drawn, by convention, to the right of the transcription
start site (+1).
•Nucleotides that lie to the left of this site (+1) are called the upstream sequences
and are identified by negative numbers.
12. RNA is transcribed from one DNA strand (the template).
In most organisms, each gene is transcribed from a single DNA strand, but different
genes may be transcribed from one or the other of the two DNA strands.
13. Polycistronic transcription in Prokaryotes
genes (operon) controlled
by one promoter and
transcribed as polycistronic
mRNA and encode multiple
15. Monocistronic transcription in Eukaryotes
3’ polyA tail
21. Requirements ofRequirements of Transcription
1.DNA Template strand (reading in 3’ → 5’ direction)
2.Enzyme: RNA Polymerase adding ribonucleoside triphosphates
(rNTPs) : ATP, GTP, CTP, UTP in the 5’ →3’ direction
3.No primer is required
22. Prokaryotic RNA polymerase
• Prokaryotes typically possess only one type of RNA polymerase, which
catalyzes the synthesis of all classes of bacterial RNA: mRNA, tRNA, and
• Bacterial RNA polymerase is a large, multimeric enzyme (meaning that it
consists of several polypeptide chains).
• The prokaryotic RNA polymerase enzyme is made up of 5 subunits
• The subunits are named α (there are two of these), β, β’, and σ. Each of
the subunits has its own job to do in transcription.
• The role of the sigma (σ) factor or subunit is to recognize a specific DNA
sequence called the promoter, which lies just upstream of the gene to be
transcribed. Without sigma, RNA polymerase will initiate transcription at
a random point along the DNA.
• E. coli promoters contain two important regions. One centered around
nucleotide −10 usually has the sequence TATATT. This sequence is called
the −10 box (or the TATA box).
• The second, centered near nucleotide −35 often has the sequence
TTGACA. This is the −35 box.
25. Prokaryotic RNA polymerase
• On binding to the promoter sequence, the σ factor brings the other subunits (two of α
and one each of β and β’) of RNA polymerase into contact with the DNA to be
transcribed. This forms the closed promoter complex.
• The two α subunits are important as they help RNA polymerase to assemble on the
• The β subunit of RNA polymerase binds rNTP and joins them together by catalyzing
the formation of phosphodiester links as it moves along the DNA template.
• The β’ subunit helps to keep the RNA polymerase attached to DNA.
• For transcription to begin, the two strands of DNA must separate, enabling one
strand to act as the template for the synthesis of an RNA molecule. This formation is
called the open promoter complex.
• There are only two hydrogen bonds between A and T ; thus it is relatively easy to
separate the two strands at this point of the promoter region.
• DNA unwinds and rewinds as RNA polymerase advances along the double helix,
synthesizing an RNA chain as it goes.
• This produces a transcription bubble. The RNA chain grows in the 5’ →3’ direction,
and the template strand is read in the 3’ →5’ direction.
• When the RNA chain is about 10 bases long, the σ factor is released from RNA
polymerase and plays no further role in transcription.
27. • RNA polymerase has to know when it has reached the end of a gene.
• Escherichia coli has specific sequences, called terminators, at the ends of
its genes that cause RNA polymerase to stop transcribing DNA.
• A terminator sequence consists of two GC rich regions that are
separated by about 10 bp. This sequence is followed by a stretch of A
• When the GC-rich regions are transcribed, a hairpin loop forms in the
RNA with the first and second GC-rich regions aligning and pairing
• Formation of this hairpin loop within the RNA molecule causes the
transcription bubble to shrink because where the template DNA strand
can no longer bind to the RNA molecule it reconnects to its sister DNA
• The RNA molecule is then released, transcription terminates, and the
double helix reforms.
• This type of transcription termination is known as rho-independent
• Some E. coli genes contain different terminator sites. These are recognized
by a protein, known as rho, which frees the RNA from the DNA. In this
case transcription is terminated by a process known as rho-dependent
Termination of prokaryotic transcription
29. Transcription in Eukaryotes
• Eukaryotes have three types of RNA polymerase.
• In eukaryotic cells, DNA is complexed with histone proteins in highly compressed
• Before transcription, the chromatin structure is modified so that the DNA is in a
more open configuration and is more accessible to the transcription machinery.
• Several types of proteins have roles in chromatin modification.
• Acetyltransferases add acetyl groups to amino acids at the ends of the histone
proteins, which destabilizes the nucleosome structure and makes the DNA more
• In addition, proteins called chromatin- remodeling proteins may bind to the
chromatin and displace nucleosomes from promoters and other regions important
30. Transcription in eukaryotesTranscription in eukaryotes
• The interaction of RNA polymerase with its promoter is far more complex in eukaryotes than it
is in bacteria.
• RNA polymerase II cannot recognize a promoter sequence. Instead, other proteins known as
transcription factors bind to the promoter and guide RNA polymerase II to the beginning of the
gene to be transcribed.
• A promoter for a gene transcribed by RNA polymerase II typically consists of two primary
parts: the core promoter and the regulatory promoter.
• The core promoter is located immediately upstream of the genecore promoter is located immediately upstream of the gene and typically contains AT-rich
sequence about 25 -30 bp upstream of the transcription start site. This sequence, called the
TATA box, binds a protein called the transcription factor IID (TFIID), one of whose subunits is
called the TATA-binding protein, or TBP.
• Mutations in the sequence of the TATA box affect the rate of transcription, and changing its
position alters the location of the transcription start site.
• Several other transcription factors (TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH) then bind to
TFIID and to the promoter region.
• TFIIF is the protein that guides RNA polymerase II to the beginning of the gene to be
• The complex formed between the TATA box, TFIID, the other transcription factors, and RNA
polymerase is known as the transcription pre-initiation complex.
• The regulatory promoter is located immediately upstream of the core promoter. Some
transcriptional activator proteins bind to regulatory promoter and, either directly or indirectly
affects the rate at which transcription is initiated.
• Some regulatory promoters also contain repressing (suppressing) sequences, which are bound
by proteins that lower the rate of transcription through inhibitory inactions with the mediator.
32. Transcription in Eukaryotes
• Enhancers are DNA sequences that increase the rate of transcription at
distant genes. The precise position of an enhancer relative to a gene’s
transcriptional start site is not critical; most enhancers can stimulate any
promoter in their neighborhood. An enhancer may be upstream or
downstream from the affected gene or, in some cases, within an intron of
the gene itself. Enhancers also contain sequences that are recognized by
transcriptional activator proteins.
• Transcription in Eukaryotes begins when the carboxy-terminal domain
of RNA polymerase II is phosphorylated. This region is rich in the amino
acids serine and threonine each of which contains an OH group in their
side chain. When these OH groups are phosphorylated, RNA polymerase II
breaks away from the pre-initiation complex and proceeds to transcribe
DNA into mRNA.
34. Transcription apparatus. The TATA-binding protein (TBP), a component of TFIID, binds toTranscription apparatus. The TATA-binding protein (TBP), a component of TFIID, binds to
the TATA box. Transcription factors TFII A and B bind to TBP. RNA polymerase binds,the TATA box. Transcription factors TFII A and B bind to TBP. RNA polymerase binds,
then TFII E, F, and H bind. This complex can transcribe at a basal level. Some coactivatorthen TFII E, F, and H bind. This complex can transcribe at a basal level. Some coactivator
proteins are present as a component of TFIID, and these can bind to other regulatory DNAproteins are present as a component of TFIID, and these can bind to other regulatory DNA
binding proteins (called specific transcription factors ortranscriptional activators).binding proteins (called specific transcription factors ortranscriptional activators).
36. A schematic view of a eukaryotic gene.. The gene consists of promoter and transcribedA schematic view of a eukaryotic gene.. The gene consists of promoter and transcribed
regions. The transcribed region contains introns, and exons. The first RNA formregions. The transcribed region contains introns, and exons. The first RNA form
produced is heterogenous nuclear RNA (hn RNA) or premature RNA or primaryproduced is heterogenous nuclear RNA (hn RNA) or premature RNA or primary
transcript, which contains both intronic and exonic sequences. The hnRNA is modifiedtranscript, which contains both intronic and exonic sequences. The hnRNA is modified
such that a cap is added at the 5 end (cap site), and a poly-A tail added to the 3 end.such that a cap is added at the 5 end (cap site), and a poly-A tail added to the 3 end.
The introns are removed (a process called splicing) to produce the mature mRNA orThe introns are removed (a process called splicing) to produce the mature mRNA or
final transcript , which leaves the nucleus to direct protein synthesis in the cytoplasm.final transcript , which leaves the nucleus to direct protein synthesis in the cytoplasm.
Py is pyrimidine (C or T).Py is pyrimidine (C or T).
37. Messenger RNA Processing: 5’ capping.
• A newly synthesized
eukaryotic mRNA (pre
mRNA) undergoes several
modifications before it leaves
• The first is known as 5’
capping. Very early in
transcription the 5-terminal
triphosphate group is modified
by the addition of a guanosine
via a 5’-5’-phosphodiester
link (triphosphate linkage) .
The guanosine is subsequently
methylated to form the 7-
methyl guanosine cap.
39. Messenger RNA Processing: poly-A tail
• The 3’ ends of nearly all eukaryotic mRNAs are modified by the addition of a long
stretch of adenosine residues, the poly-A tail .
• A sequence AAUAAA is found in most eukaryotic mRNAs about 20 bases from
where the poly-A tail is to be added and is probably a signal for the enzyme poly-A
polymerase to bind and to begin the polyadenylation process.
• The length of the poly-A tail varies, it can be as long as 250 nucleotides. Unlike
DNA, RNA is an unstable molecule, and the capping of eukaryotic mRNAs at their
5 ends and the addition of a poly-A tail to their 3 end increases the lifetime of
mRNA molecules by protecting them from digestion by nucleases.
40. Messenger RNA Processing: Exons /Introns splicing
• Eukaryotic protein-coding genes are split into exon and intron sequences. Both the
exons and introns are transcribed into mRNA.
• The introns have to be removed and the exons joined together by a process known as
RNA splicing before the mRNA can be used to make protein.
• RNA splicing takes place within the nucleus.
• Within an mRNA the first two bases following an exon are always GU and the last
two bases of the intron are AG.
• Several small nuclear RNAs (snRNAs) are involved in splicing. These are
complexed with a number of proteins to form a structure known as the spliceosome.
• One of the snRNAs is complementary in sequence to either end of the intron
sequence. It is thought that binding of this snRNA to the intron, by complementary
base pairing, brings the two exon sequences together, which causes the intron to loop
• The proteins in the spliceosome remove the intron and join the exons together.
Splicing is the final modification made to the pre mRNA in the nucleus. The mRNA
is now transported to the cytoplasm for protein synthesis.
• Exception: As well as removing introns, splicing can sometimes remove exons in a
process called alternative splicing. This allows the same gene to give rise to
different proteins at different times or in different cells.
• For example, alternative splicing of the gene for the molecular motor dynein
produces motors that transport different types of cargo