Molecular Biology: Transcription

Transcription

• RNA synthesis is the transfer of information from DNA to RNA, a template-directed process that takes place in 5' to 3' direction by polymerization of nucleoside triphosphates (NTP).
• The basic mechanism of RNA synthesis is the same for all types of RNAs: messenger (mRNAs), ribosomal (rRNAs), and transfer (tRNAs).
• RNA synthesis is catalyzed by a group of enzymes termed RNA polymerases (RNAP).

Principles of Transcription and Terminology

• Only one strand of DNA is transcribed, known as the Template strand, while the other strand is known as the Coding strand.
• The sequence of RNA is complementary to the sequence of the template strand, and it is the same as that of the coding strand except for U replacing T.
• Upstream: In the 3’ direction of the template strand.
• Downstream: In the 5’ direction of the template strand.
• Transcription region: The nucleotide sequence transcribed by RNAP.
• Promoter: A nucleotide sequence in the template strand at which the RNAP begins attachment.
• Terminator: A nucleotide sequence in the template strand that lies downstream the transcription unit and marks its end.
• Start Point: The nucleotide at the 3’ end of the transcription region.
• Transcription unit includes: The Promoter, Transcription Region, and the Terminator.

Phases of Transcription

• Template Binding & Formation of Preinitiation Complex (PIC): The binding of RNA polymerase (RNAP) to a region on the DNA of the template strand which is specific and is known as the promoter region.
• Initiation: After the promoter region is recognized by the RNA polymerase, it moves along the template strand towards the +1 nucleotide and starts to synthesize a complementary transcript of the template DNA strand in the 5’ to 3’ direction.
• Elongation: The process of elongation of the RNA chain continues until a termination signal is reached.
• Termination: When a termination signal is reached, termination occurs.

Transcription in Prokaryotes

• Prokaryotic RNA polymerase consists of a core enzyme and a sigma factor (s subunit).
• The core enzyme is formed of five subunits: 1w, 2a, 1ß, and 1ß’, responsible for the 5’ to 3’ RNA polymerase activity.
• The sigma factor enables RNA polymerase to recognize the specific promoter regions on the DNA.
• The prokaryotic promoter contains two characteristic consensus sequences: -10 element and – 35 element.
• The -10 element is formed of 6 nucleotides (5'-TATAAT-3') centered about ten nucleotides to the left of the transcription start site.
• The -35 element is a second consensus sequence (5'-TTGACA-3'), centered about 35 bases to the left of the transcription start site.

Transcription in Eukaryotes

• Eukaryotic cells contain 3 Nuclear and 1 Mitochondrial RNAP.
• Mitochondrial RNAP: Transcribes all mitochondrial genes.
• Nuclear RNA polymerases are designated by Roman Numbers I, II, and III.
• RNA polymerase I: for the synthesis of 45S RNA, which is cleaved to release the mature 18S, 5.8S, and 28S rRNA.
• RNA polymerase II: for the synthesis of mRNAs, most small nuclear RNAs (snRNAs) and microRNAs (miRNAs).
• RNA polymerase III: for synthesis of tRNAs, 5S rRNA and one snRNA.

Eukaryotic Promoter

• Two types of sequence elements are present in the eukaryotic promoter: TATA Box and CAAT box regions.
• TATA Box (Hogness Box): defines where transcription is to commence along the DNA.
• CAAT box and GC box regions: determine the Frequency of Transcription.

Steps of Transcription in Eukaryotes

• Template Binding & Formation of Preinitiation Complex (PIC): requires, in addition to RNA pol II, the general transcription factors (GTFs): TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH.
• Initiation: TFIIH has a kinase activity that activates RNAP by its phosphorylation and a helicase activity that separates the two strands of DNA for initiation.
• Elongation: The RNA polymerase utilizes ribonucleoside triphosphates (ATP, GTP, CTP, and UTP), and releases pyrophosphate each time a nucleotide is added to the growing chain.
• Termination: RNAP II ends transcription when it reaches a termination signal.