RECOMBINANT DNA202

GENE EXPRESSION: TRANSCRIPTION

Dr Danisile Tembe, SLS Room 05-105, Email: TembeD@ukzn.ac.za

 THE CENTRAL DOGMA

➢ Process by which the genetic information in the cell flow from DNA to mRNA to proteins

➢ Two stages involved: Transcription and translation

 BASIC PRINCIPLES OF TRANSCRIPTION AND TRANSLATION

➢ Instructions for the synthesis of proteins comes from genes

• RNA serve as a bridge between DNA and protein synthesis
• RNA has contains Uracil not Thymine
• DNA strand has A, C, G or T
• RNA strand has A, C, G or U
TRANSCRIPTION IN PROKARYOTES
 TRANSCRIPTION

➢ Synthesis of mRNA using information encoded in DNA

• DNA strand serve as a template strand for the synthesis of RNA nucleotides (mRNA)

➢ NB: mRNA molecule will be complementary to the DNA template

• and identical to the non-template DNA
 WHICH STRAND OF THE DNA IS COPIED?

➢ DNA is double stranded, BUT, in any gene, only ONE strand is copied into RNA
 TRANSCRIPTION

➢ 3 stages of transcription:
• Initiation
• Elongation
• Termination
 TRANSCRIPTION IN PROKARYOTES: INITIATION

➢ Promoter: is a DNA sequence where the RNA Polymerase attaches & initiates the transcription
• 2 sequences
• Start point: the nucleotides where RNA polymerase begins synthesizing the mRNA

➢ RNA polymerase: Separates 2 strands of DNA & Joins together complementary RNA
nucleotides to the DNA template strand
 TRANSCRIPTION IN PROKARYOTES: INITIATION

Prokaryotic RNA Polymerases

➢ The same RNA polymerase is used in prokaryotes to transcribe all of their genes
➢ The polymerase contain five polypeptide subunits
• β, β’, α, α and 
➢ The enzyme that comprised of all five subunits is holoenzyme
 TRANSCRIPTION IN PROKARYOTES: INITIATION

➢ Each subunit has a unique function

• The β- subunits binds to the ribonucleoside triphosphate that will become part of
the new mRNA

• The β’ -subunit binds the DNA template strand

• The two α –subunits are required to assemble the polymerase on the DNA

• The  , is involved in transcription initiation – causes the polymerase to synthesize

mRNA from an appropriate initiation site
 TRANSCRIPTION IN PROKARYOTES: INITIATION

➢ At the -10 and -35 regions upstream of the start point there are 2 promoter consensus
sequence
➢  subunit attaches to the sequences for RNA synthesis to begin
➢ RNA polymerase unwinds the DNA strand and initiate transcription
➢ Once transcription has been initiated,  subunit dissociates from the polymerase
 TRANSCRIPTION IN PROKARYOTES: ELONGATION

➢ During elongation the  subunit is released from the polymerase

➢ Causing the enzyme to proceed synthesizing mRNA in the 5’ to 3’ direction

• adds RNA nucleotide to the 3’ end of the growing RNA strand
 TRANSCRIPTION IN PROKARYOTES: TERMINATION

➢ The prokaryotic polymerase needs to be instructed/signaled to dissociate from the

DNA template
➢ Set free newly made mRNA
➢ The transcribed (produced) RNA sequence serve as the termination signal
➢ Causing the RNA polymerase to detach from the DNA
➢ & release the transcript
➢ The RNA transcript does not require further modification
➢ or processing before translation
TRANSCRIPTION IN EUKARYOTES
 INITIATION

➢ Unlike the prokaryotic polymerase that can bind to a DNA template on its own,

• Eukaryotes require transcription factors, to first bind to the promoter region

• and then to help recruit the appropriate polymerase
• However, they both have a sequence similar to the - 10 sequence in prokaryotes
• TATAAA in eukaryotes and TATAAAT in prokaryotes (-10 sequence)
 INITIATION

➢ Within the promoter there is TATA box &

transcription start point
• Essential in forming the initiation transcription
complex
• The nucleotide position where the RNA
polymerase begins the synthesis of RNA
 INITIATION

➢ Transcription factors (purple) bind to the DNA

• RNA polymerase ll can only bind to the promoter
after the transcription factors are attached

➢ RNA polymerase ll is bound to transcription factors on

the DNA,
• RNA polymerase separates DNA strands & begin
the RNA synthesis
 ELONGATION

1) As RNA polymerase moves along DNA, it untwists the double helix

2) The RNA polymerase adds RNA nucleotide to the 3’ end of the growing RNA strand
3) The newly synthesized RNA molecule peels away from its DNA template strand as
transcription continues
 TERMINATION

➢ RNA polymerase ll transcribes a sequence on a DNA called polyadenylation signal

sequence
• (AAUAAA)
• Once these 6 nucleotides appears are bound to the nucleus by certain proteins
• These proteins release the pre-mRNA
• The pre-mRNA then undergoes processing
 PRE-MRNA PROCESSING IN EUKARYOTES

➢ The mechanism of transcription is similar in bacteria and eukaryotes

• The flow of genetic information within the cell differs
 PRE-MRNA PROCESSING IN EUKARYOTES

➢ Bacteria do have nuclei – therefore ➢ Eukaryotes have nuclei – therefore

nuclear membrane do not separate nuclear envelope separates transcription

bacterial DNA & mRNA from ribosomes from translation

• Causing translation of mRNA to ➢ Transcription occurs in the nucleus

begin without additional processing however the mRNA must be transported to

• Both occurs in a cytoplasm the cytoplasm for translation

 PRE-MRNA PROCESSING IN EUKARYOTES

➢ Before RNA transcript (pre-mRNA) can leave the

nucleus it undergo processing to produce final mRNA

➢ Transcription result in pre-mRNA & further pre-mRNA

processing result in the finished mRNA
• transported to the cytoplasm for translation
 PRE-MRNA PROCESSING IN EUKARYOTES

➢ The eukaryotic pre-mRNA undergoes extensive processing before it can be

translated

➢ Three steps of pre-mRNA processing:

1. Addition of stabilizing and signaling factors at the 5’ and 3’ ends of the

molecule
2. The removal of introns –mRNA splicing
3. The mRNA transcripts can be edited after it is transcribed- in rare
cases
 PRE-MRNA PROCESSING IN EUKARYOTES
1. Addition of stabilizing and signaling factors at the 5’ and 3’
ends of the molecule:
• Alteration of mRNA ends
• 5’ capping & 3’ Poly-A-Tail
➢ 5’ capping
• a 7-methylguanosine cap (form of guanine nucleotide) is
added to the 5’ end of the growing transcript
• To protects the mRNA from degradation
• During protein synthesis the factors recognize this cap to
help initiate translation
 PRE-MRNA PROCESSING IN EUKARYOTES
➢ 3’ Poly-A-Tail
• The pre-mRNA is cut and released as soon after the
polyadenylation signal appears
• At the 3’ end, an enzyme then add more adenine
nucleotides (50-250)
• This protect the pre-mRNA from degradation
• Also serve as a binding site for a protein exporting
the processed mRNA to the cytoplasm
• Poly A polymerase name of the enzyme adds 50 –
250 adenine nucleotides
 PRE-MRNA PROCESSING IN EUKARYOTES

2. Pre-mRNA splicing: The removal of introns

➢ Process where large portions of RNA transcript are removed & the remaining
portions are re-connected
• Intron: noncoding sequence within a primary transcript that is removed from
the transcript during RNA processing
• Exon: A sequence within a primary transcript that remains in the RNA after
RNA processing
➢ The pre-mRNA splicing is conducted by spliceosomes
 PRE-MRNA PROCESSING IN EUKARYOTES

➢ During pre-mRNA processing both ends of the primary transcripts are altered/modified
• This includes cutting out of introns, joining together of exons & alteration of the 5’ and 3’
end
• This stage of RNA processing is called RNA splicing

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 PRE-MRNA PROCESSING IN EUKARYOTES

How Is Pre-mRNA Splicing Carried Out?

1. Spliceosomes recognizes the 5’ and 3’

end of the intron
2. A small RNAs in the spliceosomes pair
with nucleotide of the intron
3. Small RNAs catalyzes cutting of the pre-
mRNA & joining together the exons
WATCH TRANSCRIPTION VIDEOS
https://www.youtube.com/watch?v=_Zyb8bpGMR0

https://www.youtube.com/watch?v=_Zyb8bpGMR0

https://www.youtube.com/watch?v=SMtWvDbfHLo

https://www.youtube.com/watch?v=8_f-8ISZ164

https://www.youtube.com/watch?v=gG7uCskUOrA