Lecture 5. Transcription: DNA→RNA

Transcription is the process of the RNA synthesis on the DNA template

Different Genes for Different RNAs • mRNA - Messenger RNA: Encodes amino acid sequence of a polypeptide. • tRNA - Transfer RNA: Brings amino acids to ribosome • rRNA - Ribosomal RNA: makes up the ribosome with proteins • snRNA - Small nuclear RNA: form complexes that are used in RNA processing in eukaryotes (Not found in prokaryotes) There are 4 types of RNA, each RNA is encoded by its own type of gene.

Steps of transcription: Binding of RNA-polymerase with promoter Initiation Elongation Termination

Structure of a protein-coding gene The promoter is a base-pair sequence that specifies where transcription begins The coding sequence includes coding information for the polypeptide chain specified by the gene The terminator is a sequence that specifies the end of the mRNA transcript

5’-end of RNA molecule has a triphosphate (ATP or GTP)

-35 -10 +1 5’--TTGACA-----------TATAAT-------------start Promoters are specific regions in DNA that are recognized by RNA polymerase -35 -10 +1 5’--TTGACA-----------TATAAT-------------start Start site - the first base that will be copied into RNA -10 sequence - the Pribnow box is a sequence contained within all prokaryotic promoter regions -35 sequence is a second recognition site  subunit recognizes this sequence and connects with it. The RNA polymerase then comes into contact with Pribnow box All Pribnow boxes are variants of TATAATG. It is located 5-10 bases to left (upstream) from the first base that will be copied into RNA.

RNA polymerase holoenzyme has five subunits: Two called  One called  One called  One called  The core enzyme refers to RNA polymerase () without -subunit The  subunit dissociates after initiation of transcription. Complex enzyme

RNA polymerase holoenzyme and initiation of the RNA synthesis ββ’ αα σ σ-

Open-promoter complex - localized melting of the DNA

Chain elongation The  subunit dissociates after eight bases have been polymerized The elongation of the RNA chain is carried out by the core enzyme. The  subunit dissociates after initiation The DNA helix rewinding after RNA polymerase transcribes through it The growing RNA chain dissociates from the DNA template

Elongation Notice that the transcription process creates RNA with a base sequence complementary to DNA. Genetic information is determined by the specific sequence of bases on a DNA. The transcription process creates RNA with a base sequence complementary to DNA

Termination of transcription is determined by specific sequence in the DNA The 3’-end of RNA is shown An inverted-repeat sequence (GC) can form a stem and loop configuration for RNA A sequence high in AU is near the loop (in red)

Rho (ρ)-protein must participate in chain termination The rho-protein releases the RNA polymerase and RNA molecule from DNA using ATP

The antibiotic rifampicin blocs the active site of RNA-polymerase It inhibits the chain initiation, not chain growth/ It blocs the active site of RNA-polymerase Rifampicin is the inhibitor of chain initiation

Eukaryotic cells contain three classes of RNA polymerases: RNA polymerize I (RNA pol I) is localized in the nucleolus transcribes ribosomal RNA (rRNA) genes RNA polymerize II (RNA pol II) is localized in the nucleoplasm transcribes messenger RNA (mRNA) and small nuclear RNA (snRNA) is strongly inhibited by α-amanitin (a mushroom poison) RNA polymerize III (RNA pol III) transcribes transfer RNA (tRNA) and 5S rRNA is less sensitive to α-amanitin than pol II Each class synthesizes a different type of RNA molecules and recognizes different type of promoters.

α – аmanitin is a toxin of a poison mushroom Amanita phalloides

The promoters of the eukaryotic genes are more complex The recognition sequences in promoters for RNA-polymerase II are: –25 TATA-box –50/150 CAT-box –300 GC-box

They are the specific proteins named Transcriptional Factors (TF) are necessary for the initiation of transcription in eukaryotic cells They are the specific proteins named TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH

The formation of initiation complex for RNA polymerase II The first step of initiation: TFIID binds to TATA-box Then other transcriptional factors bind to promoter to form the pre-initiation complex (PIC) TFIIH factor activates the enzyme RNA polymerase II for elongation