1. Factors Involved In RNA synthesis and processing Presented by Md. Anower Hossen ID: 10276001 MS in Biotechnology

2. RNA Synthesis and Processing Key Concepts and Learning Objectives Transcription elongation in eukaryotes is tightly coupled to RNA processing. RNA splicing removes intron sequences from newly transcribed pre-mRNAs. RNA splicing is catalyzed by the spliceosome. RNA-processing enzymes generate the 3’-end of eukaryotic mRNAs. Noncoding RNAs are also synthesized and processed in the nucleus.

3. Summary of steps from a gene to a protein Fig. 01

4. Differences in transcription of procaryotes and eucaryotes Procaryotes: transcription and translation occur in the same compartment. There are no introns to splice out and the mRNA is not capped or polyadenylated. Eucaryotes: transcription takes place in the nucleus and the primary transcript is spliced to remove introns (non-coding) and join exons (coding regions) together. The mRNA is capped and polyadenylated in the nucleus and then exported to the cytoplasm for translation.

5. 1. Transcription control of gene regulation is controlled by: 1.Promoters Occur upstream of the transcription start site. Some determine where transcription begins (e.g., TATA), whereas others determine if transcription begins. Promoters are activated by specialized transcription factor (TF) proteins (specific TFs bind specific promoters). One or many promoters (each with specific TF proteins) may occur for any given gene. Promoters may be positively or negatively regulated.

6. 1. Transcription control of gene regulation is controlled by: Cont… 2.Enhancers Occur upstream or downstream of the transcription start site. Regulatory proteins bind specific enhancer sequences; binding is determined by the DNA sequence. Loops may form in DNA bound to TFs and make contact with upstream enhancer elements. Interactions of regulatory proteins determine if transcription is activated or repressed (positively or negatively regulated).

7. More about promoters and enhancers: Some regulatory proteins are common in all cell types, others are specific. Each promoter and enhancer possesses a specific set of proteins (coactivators) that determines expression. Rate of gene expression is controlled by interaction between positive and negative regulatory proteins. Combinatorial gene regulation; enhancers and promoters bind many of the same regulatory proteins, implying lots of interaction with fine and coarse levels of control.

8. EUKARYOTIC GENE EXPRESSION A single RNA polymerase catalyzes all transcription in E. coli, but in eukaryotes there are three RNA polymerases I, II, and III, are more complex, with 10 or more subunits, than the E. coli RNA polymerase. Moreover, unlike the E. coli enzyme, all three eucaryotic RNA polymerases require the assistance of ncr proteins called transcription factors in order to initiate synthesis of RNA chains. RNA polymerase I is located in the nucleolus, where rRNAs are synthesized and combined with ribosomal proteins. RNA polymerase I catalyzes the synthesis of all ribosomal RNAs except the small 5S rRNA. RNA polymerase II transcribes nuclear genes that encode proteins and perhaps other genes specifying RNAs. RNA polymerase III catalyzes the synthesis of the transfer RNA molecules, the 5S rRNA molecules, and small nuclear RNAs.

9. Cont….. In eukaryotic cells, transcription takes place in the nucleus. The mRNA must be completely synthesized and move thorough the nuclear membrane to the cytoplasm before translation can begin. In eukaryotic cells the regions of genes that code for proteins are often interrupted by noncoding DNA. Thus eukaryotic genes are composed exons, the regions of DNA expressed, and introns, the intervening regions of DNA that do not encode proteins. In addition the RNA undergoes processing before it leaves the nucleus. The long RNA is processed by ribozymes, which removes the intron-derived-RNA amd splice together the exon-derived RNA, producing an mRNA.

10. Initiation mRNA synthesis Unlike their prokaryotic counterparts, eukaryotic RNA polymerases cannot initiate transcription by themselves. All three eukaryotic RNA polymerase require the assistance of protein transcription factors to start the synthesis of an RNA chain. these transcription factors must bind to a region in DNA and form an appropriate initiation for RNA polymerase will bind and initiate transcript promoters and transcription factors are utilize RNA polymerases I, II, and III The initiation transcription involves formation of locally unwound segment of DNA providing a DNA strand that is free to function as a template for the synthesis of a complementary strand of RNA. The formation of the locally unwound segment of DNA required to initiate transcription involves the interactions of several transcription factors with specific sequences in the promoter for the transcription unit.

11. Cont…… The promoter region lies on the upstream of the transcription unit of the gene.In many genes the promoter region contains the base sequence TATA which ensures that the transcription starts at a proper point. The RNA polymerase II binds to the promoter to open up the DNA helix and gains access to the DNA strand which is complementary to the future mRNA sequence. The promoter region confers the basal rate to transcription.

12. TFIID is the basal transcription factor to interact with the promoter, it contains a TATA binding protein and several small TBP associated proteins. Next TFIIA joins the complex followed by TFIIB. TFIIF first as sociats with RNA polymerase II and then TFIIF and RNA polymerase II join the transcription initiation complex together. TFIIF contains two subunits, one of which has DNA unwinding activity.TFIIE then joins the initiation complex, binding to the DNA downstream from the transcription start point.

13. RNA chain elongation and the addition of 5' methyl guanosine cap Once eukaryotic RNA polymerases have been released from their initiation complexes, they catalyze RNA chain elongation by the same mechanism as the RNA polymerases of prokaryotes. Early in the elongation process, the 5' ends of eukaryotic pre-mRNAs are modified by the addition of 7-methyl guanosine (7-MG) caps. These 7-MGcaps are added when the growing RNA chains are only about 30 nucleotides long. Termination by chain cleavage and the addition of 3' Poly (A) tails: Transcription proceeds beyond the site that will become the 3' terminus, and the distal segment is removed by endonucleolytic cleavage. This cleavage event produces the 3' end of a transcript usually occurs at a site 11 to 30 nucleotides downstream from a conserved sequence, consensus AAUAAA, and upstream from a G-U rich sequence located near the end of the transcription unit. After the cleavage, the enzyme poly(A) polymerase adds poly (A) tails, tracts of adenosine monophosphate residues about 200 nucleotides long, to the 3'ends of the transcripts.

15. Following transcription, the introns are removed within the nucleus from the primary mRNA molecule by a process of splicing with the help of spliceosomes, which are derived from several small ribonucleoprotein complexs. The mature mRNA thus formed is made up of exons only and enters the cytoplasm through the nuclear pores. Cont……