Lecture 3 Transcription and RNA PDF

By the end of this lecture the students will be able to: Identify different types, structure and function of RNA Know different requirement of transcription. Define DNA polymerase structure and function. Understand the different steps of transcription Discuss the different types of post transc...

By the end of this lecture the students will be able to: Identify different types, structure and function of RNA Know different requirement of transcription. Define DNA polymerase structure and function. Understand the different steps of transcription Discuss the different types of post transcriptional modifications. RNA structure  Ribosomal RNA (rRNA) Form 80% of cellular RNA THE MOST abundant form. It is synthesized inside the nucleus particularly in the nucleolus These newly synthesized rRNAs combine with ribosomal proteins and form smaller subunits and larger subunits of ribosomes respectively. Ribosomes provide necessary infrastructure for the mRNA, tRNA and amino acids to interact with each other for the translation process. Thus, ribosomal assembly is the protein synthesizing machinery. Additionally, rRNA also has enzymatic activity (peptidyl transferase) and catalyzes the formation of the peptide bond in between two aligned proteins/amino acids during protein synthesis. Transfer RNA (tRNA) They transfer amino acids from cytoplasm to the ribosomal protein synthesizing machinery; hence the name transfer RNA. tRNA molecules vary in length from 74 to 95nucleotides, having high content of unusual and modified bases. The tRNAs act as adapter molecules between mRNA and the amino acids coded by it. The tRNA molecule shows specificity in both aspects; in recognizing the mRNA codon as well as in accepting the specific amino acid coded by that codon. There are at least 20 of tRNA molecules in every cell, at least one corresponding to each of the 20 amino acids required for protein synthesis. precursor mRNA (pre-mRNA) Which of the following RNAs are the most abundant in an animal cell? (a) mRNA (b) tRNA (c) miRNA (d) rRNA Prokaryotic RNA polymerase is consisted of 5 subunits (2 α, β, β`, ω) called together core enzymes + a movable sigma (σ) subunit. The six subunits together named holoenzyme sigma (σ) recognizes the promoter region. CORE ENZYME HOLOENZYME The synthesis of a DNA can be divided into three stages I. Initiations: RNA polymerase recognizes a promoter site II. Elongation synthesizing a complementary copy of the DNA template III. Termination polymerase encounters a termination sequence it releases the RNA  Transcription Initiation Site “Upstream” “Downstream” -5 -4 -3 -2 -1 +1 +2 +3 +4 +5 +6 5’ 3’ 3’ 5’ Direction of transcription Template strand, non- coding strand There is no “zero” The promoter: It is region of the DNA, which lies upstream (left side) of the +1 start site of transcription. Characterized by the presence of the -10 region or Pribnow box and -35 region 1-Initiation  Initiation starts by recognition and binding of the RNA polymerase to the promoter region.  Polymerase hollo enzyme binds non- specifically to DNA.  Sigma subunit helps the enzyme to find the promoter area by a its sigma subunit  ------  Once finding the promoter area, the Initiation enzyme binds firmly forming closed initiation complex.  Unwinding of DNA forming opened initiation comple (transcription bubble )  Initiate of mRNA synthesis almost always with purine nucleotides.  2- Recognition and initiation begins Elongation with purines sigma subunit leaves the core enzyme after the transcription of 8 Say goodbye to bases and the core enzyme continues the sigma subunit transcription. Elongation of RNA chain is continuous  RNA polymerase DNA Promoter mRNA mRNA  3- Termination separation of the newly synthesized RNA from its DNA template 1- - (rho) dependent termination. -is protein factor. ATP is needed 2- - (rho) independent termination depends on DNA sequence called palindromic sequence Pribnow box in the promoter region of transcription is located at 1- (-10) upstream of the starting point of transcription 2- (+10) upstream of the starting point of transcription 3- (-10) downstream of the starting point of transcription 4- (+10) downstream of the starting point of transcription TATA or Hogness box CAAT box GC rich regions Post-transcriptional Modification of RNA  Post-transcriptional Modification of RNA Definition  The newly synthesized RNA molecule is called primary transcript. That are extensively modified Post-transcriptionally  A- Processing of eukaryotic and prokaryotic ribosomal RNA (splicing) Prokaryotic and eukaryotic ribosomal RNAs are synthesized from pre-ribosomal RNAs. Preribosomal RNAs are cleaved to yield intermediate sized pieces of rRNA which are trimmed to produce the required ribosomal RNA species. a cloverleaf having three loops and an open end. are usually 75-90 ribonucleotides in length. The three-loop consisting arms are namely: 1- DHU or D arm contains the base dihydrouridine, which has recognition site for specific enzyme amino-acyl tRNA synthetase 2- T arm (TΨC) (ribothymidine, pseudouridine and cytidine residues) that consists of ribosome recognition site 3- Anticodon arm that recognizes and bind to mRNA present in the ribosome. The open end with no loop is the site for attachment of amino acid, via 3’ OH bonding with COOH- group of the amino acid.  Processing of mRNA Processing of mRNA in prokaryotes eukaryotes primary transcripts of mRNA are mRNA is synthesized by RNA Pol II as subject to little processing before longer precursors called translation, this is because the site of heterogeneous nuclear RNA (hnRNA) transcription is not compartmentalized into nucleus as in Among hnRNA, those processed to eukaryotes. give mature mRNAs are called pre- mRNAs It can start to be translated before it Pre-mRNA molecules are processed to has finished being transcribed. mature mRNAs by 5’-capping, 3’- Prokaryotic mRNA is degraded rapidly cleavage and polyadenylation, splicing from the 5’ end. and methylation. Processing occurs primarily within the nucleus.  1. Capping of 5’ end by GTP 7-methylguanosine is linked to the 5` terminal residue of mRNA through unusual 5 ` -5 ` triphosphates linkage. FUNCTION: The 5`cap permits the initiation of translation and helps stabilize the mRNA and Stimulate splicing of first exon  2- Addition of a poly-tail At the 3` end of mRNA, a tail of 40-200 adenylate residues This poly A-tail : helps to stabilize the mRNA. Splicing of last intron Increased Increase translational efficiency  3- Splicing Removal of introns (intervening sequence that do not code for amino acid- and joining of exons (amino acid coding sequence).  Spliceosome  Spliceosome:- Is a special structure, involved in converting the primary transcript into mature mRNA. It consists of : 1- The primary mRNA transcript. 2- 5 small muclear RNAs (U1, U2, U4 , U5 and U6). 3- More than 60 polypeptides. Collectively, they form a nucleoprotein complex sometimes called "snurp".  ALTERNATIVE mRNA PROCESSING Alternative splicing The generation of different mature mRNAs from a particular type of gene transcript can occur by varying the use of 5’- and 3’- splice sites in different ways: (i) By retaining certain introns. (ii) By retaining or removing certain exons 4- RNA editing: An unusual form of RNA processing in which the sequence of the primary transcript is altered is called RNA editing. Changing RNA sequence (after transcription).  5- Pre-mRNA methylation The final modification or processing event that many pre-mRNAs undergo is specific methylation of certain bases to regulates gene expression. The methylations seem to be largely conserved in the mature mRNA. Secondary methylation of the mRNA molecules, on the 2'-hydroxy and the N6 of adenylyl residues, occur after the mRNA molecule has appeared in the cytoplasm.  Reverse Transcription Normal transcription involves synthesis of RNA from DNA. Reverse transcription is the transcription of single stranded RNA into double stranded DNA With the help of the enzyme Reverse Transcriptase.  Retrovirus Replication Cycle ds DNA host DNA Provirus Packaging into virus ; budding  Which of the following best describes the 'cap' modification of eukaryotic mRNA? 1- A modified guanine nucleotide added to the 3' end of the transcript. 2- A modified guanine nucleotide added to the 5' end of the transcript. 3- A string of adenine nucleotides added to the 3' end of the transcript. 4- A string of adenine nucleotides added to the 5' end of the transcript.  Reference  Harvey, Richard A., Ph. D. (2017). Lippincott's illustrated reviews: Biochemistry. Philadelphia :Wolters Kluwer Health, 7th edition