RNA Synthesis (Transcription) PDF

**RNA SYNTHESIS (TRANSCRIPTION)** ***CONTENTS*:** I. **Introduction** II. Transcription Phases III. Transcription in Prokaryotes IV. Transcription in Eukaryotes V. Post-transcriptional modifications of messenger RNA **RNA SYNTHESIS (TRANSCRIPTION)** **Definition:** Transfer of information...

**RNA SYNTHESIS (TRANSCRIPTION)** ***CONTENTS*:** I. **Introduction** II. Transcription Phases III. Transcription in Prokaryotes IV. Transcription in Eukaryotes V. Post-transcriptional modifications of messenger RNA **RNA SYNTHESIS (TRANSCRIPTION)** **Definition:** Transfer of information from DNA to RNA **Terminology** - **Upstream**: the 3' direction of the template strand - **Downstream:** the 5' direction of the template strand. - **Transcription region:** Nucleotide sequence on the template strand transcribed by the RNA polymerase. - **Promoter:** Nucleotide sequence on template strand upstream the transcription unit, where the RNAP begins attachment to DNA. - **Terminator:** Nucleotide sequence on template strand downstream the transcription unit, marking the end of transcription. - **The start point +1:** The nucleotide at the 3' end of transcription region. - **Positive numbers:** increase as we go downstream the transcription unit. - **Negative numbers:** A base in the promoter region is assigned a negative number if it occurs prior (to the left, or upstream) to the transcription start site. - **Transcription unit:** includes the promoter, transcription region and the terminator. - **Element or Box:** Any defined nucleotide sequence and repeated in DNA. **Principals of Transcription** 1. Only one strand of DNA is transcribed. **The strand of DNA that is transcribed into RNA is** known as **the template strand** whereas **the other strand is** known as the **coding strand**. 2. The sequence of RNA is complementary to the sequence of template strand and it is the same as that of coding strand except for **U replacing T**. 3. RNA synthesis includes the following **main phases**: a. b. c. d. **Transcription in Prokaryotes** 1. There is **a single DNA dependent RNA polymerase in prokaryotes**. It catalyzes the synthesis of all types of RNAs. It catalyzes the polymerization of ribonucleoside triphosphates as directed by DNA template in 5\' → 3\' direction (it reads the template in 3\' → 5\' direction). Unlike DNA polymerases it does not require primer to initiate polymerization as it does not have proofreading activity. 2. **Prokaryotic RNA Polymerase** consists of: a. **[Core enzyme]** which is formed of **five subunits:1ω, 2α, 1β, and 1β\'.** It cannot recognize the promoter region on the DNA template. b. **[The σ subunit]** (sigma factor): enables RNA polymerase to recognize the specific promoter regions on the DNA. Different sigma factors help recognition of different promoters. It is released after initiation of transcription, The core enzyme + the sigma factor= **holoenzyme** 3. **Termination of transcription**: **Clinical application: Prokaryotic RNA Polymerase** is inhibited by rifampicin. Actinomycin D binds to the DNA preventing transcription. **[The Prokaryotic promoter:]** The prokaryotic promoter contains characteristic consensus sequences. A consensus sequence in the genome is a specific sequence of nucleotides that plays the same role in different locations. **These consensus sequences are** **recognized by prokaryotic RNA polymerase- σ** complex and they include: 1. 2. **Transcription in Eukaryotes** The chemistry of transcription in eukaryotes is the same General Principles Types of RNA Polymerases 1. 2. 3. **[Eukaryotic promoter]** Two types of sequence elements are present in eukaryotic promoter (**basal expression elements).** 1. 2. **[Steps of Transcription]** **[I- Template Binding & Formation of Preinitiation Complex (PIC):]** - This step requires, in addition to pol II, the **(GTFs): TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH.** - **TFIID,** which **binds to the TATA box promoter element,**is the only one of these factors that is independently capable of specific high affinity binding to promoter DNA. - This is followed by binding of the other transcription factors, including TFIIF, which brings with it RNAP. **[II- Initiation]** - **TFIIH** has a **kinase activity that activates RNAP** by its phosphorylation and a **helicase activity** and separates the two strands of DNA for initiation. - This is followed by release of TFII A, B, E & H. Then, **Pol II-TFIIF** complex leaves the promoter region,moves towards the +1 nucleotide and starts to synthesize a complementary transcript of the template DNA strand. **[III- Elongation]** **[IV- Termination ]** RNAP II ends transcription when it reaches a **termination signal**. Transcription proceeds till the termination consensus sequence **AAUAAA**is reached. **Post-transcriptional Modifications** All three types of RNAs are synthesized in precursor forms in eukaryotes. These precursors are converted to functional RNA molecules by post-transcriptional modifications. **[I- Post-transcriptional modificationsof messenger RNA]** - Messenger RNA is synthesized as a primary transcript known as heterogeneous nuclear RNA (hnRNA). - Post transcriptional modifications include mainly: **[1-Capping at 5′ end]** **Methyl-guanosine cap** is added at the 5\' end. **Importance of capping:** a. b. **[2- Polyadenylation at 3′ end]** **Poly (A) polymerase (PAP)** is responsible for the addition of poly(A) tail at the 3\`end of pre-mRNA. a. **[3- Splicing]** - Parts of hnRNA do not code for amino acids. These sequences are known as **introns**. They lie between amino acid coding regions known as **exons**. - Splicing removes introns (nonexpressed regions) and joins exons(expressed regions) to form functional mRNA. - Splicing requires a **splicesome** formed of special proteins and small nuclear RNAs (snRNAs). Usually snRNAs consist of about 100 nucleotides. The base composition of snRNA is complementary to the ends of introns. This helps snRNA and introns to base pair and this leads to loop formation. - As a result, adjacent exons come together. - Finally, removal of intron loop and joining of exons takes place. **Alternative splicing:** - -The primary transcript of some genes may be spliced differently to yield different proteins from the same gene. - **Clinical application:** 1. **Systemic Lupus erythematosus (SLE)**is an autoimmune disease characterized by autoantibodies against many cellular components. Small nuclear ribonucleoproteins (snRNPs) are one of the targets of these antibodies. 2. **One type of β-thalassemia** results from a nucleotide change at the intron-exon junction leading to failure to remove the introns and hence decreased synthesis of the β- globin chain. **[4- mRNA editing]** Coding information of mRNA can be changed by RNA editing. **For example:** - **In the liver**: the single apolipoprotein gene (apoB gene) is transcribed into an mRNA that directs the synthesis of **apoB-100 protein** (100-kDa). - **In the intestine**: the same gene directs the synthesis of a primary transcript, then by the action of a cytidine deaminase, a **CAA** codon in mRNA is converted to **UAA** which is a termination codon. **Apo B-48 protein** (48-kDa) is the result of translating this edited form of mRNA. **ApoB-100 and Apo B-48** proteins have different functions.