The document discusses the process of transcription in prokaryotes and eukaryotes. It describes the three main stages of transcription - initiation, elongation, and termination - and how they differ between prokaryotes and eukaryotes. In eukaryotes, the mRNA transcript undergoes processing including capping, polyadenylation, and splicing before being exported from the nucleus, while in prokaryotes the mRNA is used directly for translation. The structures of RNA polymerases also differ between the two systems.

1. Transcription
Synthesis of a single-stranded RNA molecule using the
DNA template (1 strand of DNA is transcribed)
Aman Ullah
B.Sc. Med. Lab. Technology
M. Phil. Microbiology
Certificate in Health Professional Education
Lecturer, Department of Medical Lab.
Technology
Institute of Paramedical Sciences, Khyber
Medical University, Peshawar, Pakistan

2. Gene
Unit of DNA that contains the information to
specify synthesis of a single polypeptide chain or
functional RNA (such as a tRNA)

3. Transcription
• Synthesis of RNA, the four-base language of DNA
containing A, G, C, and T is simply copied, or
transcribed, into the four-base language of RNA,
which is identical except that U replaces T
• In this lecture we focus on formation of
functional mRNAs from protein-coding genes
• A similar process yields the precursors of rRNAs
and tRNAs encoded by rRNA and tRNA genes;
these precursors are then further modified to
yield functional rRNAs and tRNAs

4. Transcription
• During transcription of DNA, one DNA strand acts as a
template, determining the order in which ribonucleoside
triphosphate (rNTP) monomers are polymerized to form a
complementary RNA chain
• Bases in the template DNA strand base-pair with
complementary incoming rNTPs, which then are joined in a
polymerization reaction catalyzed by RNA polymerase
• Polymerization involves a nucleophilic attack by the 3
oxygen in the growing RNA chain on the phosphate of the
next nucleotide precursor to be added, resulting in
formation of a phosphodiester bond
• As a consequence of this mechanism, RNA molecules are
always synthesized in the 5‘ to 3' direction

6. Transcription
• Like the two strands in DNA, the template DNA strand
and the growing RNA strand that is base-paired to it
have opposite 5‘ to 3' directionality
• The site at which RNA polymerase begins transcription
is numbered +1
• Downstream denotes the direction in which a
template DNA strand is transcribed (or mRNA
translated)
• Upstream denotes the opposite direction
• Nucleotide positions in the DNA sequence downstream
from a start site are indicated by a positive (+) sign;
those upstream, by a negative(-) sign

7. Stages in transcription
1. Initiation: RNA polymerase recognizes and
binds to a specific site, called a promoter, in
double-stranded DNA
• After binding to a promoter, RNA polymerase
melts the DNA strands in order to make the
bases in the template strand available for base
pairing with the bases of the ribonucleoside
triphosphates that it will polymerize together

8. Stages in transcription
• The enzyme maintains a melted region of
approximately 14 base pairs, called the
transcription bubble
• Transcription initiation is considered complete
when the first two ribonucleotides of an RNA
chain are linked by a phosphodiester bond

9. Promoter Transcription unit
RNA polymerase
Start point
DNA
5
3
3
5

10. Stages in transcription
2. Elongation: RNA polymerase moves along the
template DNA one base at a time, opening the
double-stranded DNA in front of its direction of
movement and hybridizing the strands behind it
• One ribonucleotide at a time is added to the 3 end of
the growing (nascent) RNA chain during strand
elongation by the polymerase
• Approximately eight nucleotides at the 3 end of the
growing RNA strand remain base-paired to the
template DNA strand in the transcription bubble

11. Elongation
Non-template
strand of DNA
RNA
polymerase
RNA nucleotides
3 end
3
5
5
Newly made
RNA
Template
strand of DNA
Direction of transcription
(“downstream”)

12. Stages in transcription
• The elongation complex, comprising RNA polymerase,
template DNA, and the growing (nascent) RNA strand,
is extraordinarily stable
3. Termination: The final stage in RNA synthesis, the
completed RNA molecule, or primary transcript, is
released
from the RNA polymerase and the polymerase
dissociates from the template DNA
• Specific sequences in the template DNA signal the
bound RNA polymerase to terminate transcription
• Once released, an RNA polymerase is free to transcribe
the same gene again or another gene

14. Promoter
3
5
Transcription unit
DNA
Initiation
RNA polymerase
Start point
Template strand
of DNA
RNA
tran-
script
Unwound
DNA
Elongation
3
3
5
3
5
5
3 5
Rewound
DNA
5 3
35 3
5
RNA
transcript Termination
35
5 3
Completed RNA transcript

15. General concepts
• Three phases: initiation, elongation,
and termination.
• The prokaryotic RNA-pol can bind to
the DNA template directly in the
transcription process.
• The eukaryotic RNA-pol requires co-
factors to bind to the DNA template
together in the transcription process.

16. mRNA differences between prokaryotes and eukaryotes:
Prokaryotes
1. mRNA transcript is mature, and used directly for translation without
modification.
2. Since prokaryotes lack a nucleus, mRNA also is translated on ribosomes before it
is transcribed completely (i.e., transcription and translation are coupled).
3. Prokaryote mRNAs are polycistronic, they contain amino acid coding information
for more than one gene.
Eukaryotes
1. mRNA transcript is not mature (pre-mRNA); must be processed.
2. Transcription and translation are not coupled (mRNA must first be exported to
the cytoplasm before translation occurs).
3. Eukaryote mRNAs are monocistronic, they contain amino acid sequences for just
one gene.

17. Structure of RNA Polymerases
• The RNA polymerases of bacteria and eukaryotic cells
are fundamentally similar in structure and function
• Bacterial RNA polymerases are composed of two
related large subunits ( β'and β), two copies of a
smaller subunit (α), and one copy of a fifth subunit (ω)
that is not essential for transcription or cell viability but
stabilizes the enzyme and assists in the assembly of its
subunits
• Eukaryotic RNA polymerases have several additional
small subunits associated with this core complex

18. Prokaryotes possess only one type of RNA polymerase
 transcribes mRNAs, tRNAs, and rRNAs
Eukaryotes possess three RNA polymerases:
1. RNA polymerase I, transcribes three major rRNAs 12S, 18S, 5.8S
2. RNA polymerase II, transcribes mRNAs and some snRNAs
3. RNA polymerase III, transcribes tRNAs, 5S rRNA, and snRNAs

19. Cells Produce Several Types of RNA
• The majority of genes carried in a cell’s DNA specify the
amino acid sequence of proteins; the RNA molecules that
are copied from these genes (which ultimately direct the
synthesis of proteins) are called messenger RNA (mRNA)
molecules
• The final product of a minority of genes, however, is the
RNA itself
• These RNAs, like proteins, serve as enzymatic and structural
components for a wide variety of processes in the cell
• Ribosomal RNA (rRNA) molecules form the core of
ribosomes
• Transfer RNA (tRNA) molecules form the adaptors that
select amino acids and hold them in place on a ribosome
for incorporation into protein

20. Three Steps to Transcription:
1. Initiation
2. Elongation
3. Termination
 Occur in both prokaryotes and eukaryotes
 Elongation is conserved in prokaryotes and eukaryotes
 Initiation and termination proceed differently

21. Termination of Transcription
Different in prokaryotes and eukaryotes
• In prokaryotes
• RNA pol stops transcription at the end of the
terminator (DNA sequence)
• In eukaryotes
• pre-mRNA is cleaved from the growing RNA chain
• RNA pol eventually falls off the DNA

22. RNA processing in eukaryotes, not prokaryotes
1. Addition of methylated cap to 5’ end of messenger RNA (mRNA)->
increases stability and translation of mRNA
2. Addition of poly(A) tail to 3’ end (polyadenylation) -> increases stability and
translation of mRNA
3. Splicing
removal of introns and joining together of exons
All processing events occur in nucleus
before transport to cytoplasm

23. 5 Exon Intron Exon Intron Exon 3
Pre-mRNA
1 30 31 104 105 146
Coding
segment
Introns cut out and
exons spliced together
1 146
5 Cap
5 Cap
Poly-A tail
Poly-A tail
5 3UTR UTR
(mature) mRNA

24. Termination in Prokaryotes, E. coli model:
Two types of terminator sequences occur in prokaryotes:
1. Type I (-independent)
Palindromic, inverse repeat forms a hairpin loop and is believed to physically
destabilize the DNA-RNA hybrid.
2. Type II (-dependent)
Involves  factor proteins that break the hydrogen bonds between the template
DNA and RNA.

25. Questions/Suggestions
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