RNA Synthesis and Processing

RNA Synthesis and ProcessingLecture 3Objectives:1. To understand the transcription process2. To understand how RNA polymerase generate a new single-stranded RNA3. To define prmotors and promoter sequence4. To describe RNA processing eukaryotic cellsTranscriptionThe process of RNA synthesis directed by a DNA template is termed transcription, and occursin three phases: initiation, elongation and termination.In transcription, DNA is copied to RNA by an enzyme called RNA polymerase .Transcription to yield an mRNA is the first step of protein biosynthesis .1. initiation of transcriptioni. Promoter sequences. Unlike the initiation of replication, transcriptional initiation doesnot require a primer. Promoter sequences are responsible for directing RNApolymerase to initiate transcription at a particular point. Promoter sequences differbetween prokaryotes and eukaryotesIn genetics, a promoter is a DNA sequence that enables a gene to be transcribed. Thepromoter is recognized by RNA polymerase (RNAP), which then initiates transcription.1. Prokaryotic promoters. The promoters for most prokaryotic genes have threesequence elements.a. Initiation site (startpoint). Transcription for most genes always starts at thesame base (position one). The startpoint is usually purine.b. Pribnow box. : lies 9-18 base pairs upstream of the startpoint.i. Its either identical to or very similar to the sequence TATAAT.ii.The pribnow box also called -10 sequence because it is usually found10 bp upstream of the startpoint.c. The -35 sequence is a component of a typical prokaryotic promoter. It is aTTGACA. Called -35 sequence because it is usually found 35bp upstream ofthe startpoint.2. Eukaryotic Promoters. Each type of eukaryotic RNA polymerase uses a differentpromoter. The promoters used by RNA polymerase I and II are similar to theprokaryotic promoter in that they are upstream of the startpoint. However, thepromoters used by RNA polymerase III are unique because they are usuallydownstream of the startpoint.2013 1 Professor H.D.El-Yassin

Lecture 3ii. Initiation factors:1. Prokaryotic σ factor is required for accurate initiation of transcription.2. Eukaryotic initiation factors: the initiation of transcription in eukaryotes isconsiderably more complex than in prokaryotes, partly because of the increasedcomplexity of eukaryotic RNA polymerases and partly because of the diversity oftheir promoters.2. Elongation:The basic requirement and fundamental mechanism of the elongation phase of RNA synthesisis the same in prokaryotes and eukaryotes.1) Template: A single strand of DNA acts as a template to direct the formation ofcomplementary RNA during transcription.2) Substrates: the four nucleosides triphosphates are needed as substrates for RNAsynthesis.3) Direction of synthesis: RNA chain growth proceeds in the 5' to 3' direction.4) Enzyme:a. Prokaryotes have a single RNA polymerase responsible for all cellular synthesis.Thestructure of RNA polymerase is complex:b. Eukaryotes have one mitochondrial and three nuclear RNA polymerase. The latterare distinct enzymes that function to synthesize different RNAs.3. Termination:i. In prokaryotices:There are two basic classes of termination event in prokaryotes1. Intrinsic termination (Rho-independent termination) involves terminator sequences within theRNA as it is being made that signal the RNA polymerase to stop. The terminator sequenceis usually a palindromic DNA sequence that forms a hairpin.2. Rho-dependent termination uses a termination factor called ρ factor to stop RNA synthesisat specific sites. When ρ-factor reaches the RNAP, it causes RNAP to dissociate from theDNA, terminating transcription.2013 2 Professor H.D.El-Yassin

Lecture 3ii. In eukaryotices: Very little is known about how they terminate transcription2013 3 Professor H.D.El-Yassin

Posttranscriptional RNA processingLecture 3Once a gene transcript has been synthesized, numerous post-transcriptional modification orprocessing events may be needed before the transcript is functional.1. Prokaryotes: post-transcriptional processing of RNA is not as extensive in prokaryotesas in eukaryotes; however, some processing does occur.2. Eukaryotes: Overall, post-transcriptional processing is more extensive in eukaryotesthan in prokaryotes. This partly is due to the presence of a nucleus from which mostRNAs must be transported. RNAs are processed during this transport. Processing givesthem the characteristics they need to be functional in the cytoplasm such as anincreased stability of mRNAs as well as allowing for another level of gene regulation.a. The primary transcript (hnRNA) is capped at its 5' end as it is being transcribed.b. A poly (A) tail, 20 to 200 nucleotide in length is being added to the 3' end of hetranscript.c. Splicing reactions remove introns and connect the exons.(The most common cause of -thalassemia are defects in mRNA splicing of the -globin gene.Mutations that affect the splicing create aberrant transcript that are degraded before they aretranslated. If patients inherit a single mutant gene thalassemia minor, the disease manifestsitself with a mild anemia. However, patents with homozygous mutations thalassemia majorhave sever transfusion-dependent anemia.2013 4 Professor H.D.El-Yassin

2013 5 Professor H.D.El-YassinLecture 3

Lecture 3Conclusion:1. Synthesis of RNA from a DNA template is called transcription2. Genes are transcribed by enzymes called RNA polymerases that generate a singlestrandedRNA identical in sequence (with the exception of U in place of T) to one of thestrands of the double-stranded DNA. The DNA strand that directs the sequence ofnucleotides in the RNA by complementary base-pairing is the template strand. TheRNA strand that is initially generated is the primary transcript. The DNA template iscopied in the 3' to 5' direction, and the RNA transcript is synthesized in the 5' to 3'direction. RNA polymerases differ from DNA polymerases in that they can initiate thesynthesis of new strands in the absence of a primer.3. In addition to catalyzing the polymerization of ribonucleotides, RNA polymerases mustbe able to recognize the appropriate gene to transcribe, the appropriate strand of thedouble-stranded DNA to copy, and the startpoint of transcription . Specific sequenceson DNA, called promoters, determine where the RNA polymerase binds and howfrequently it initiates transcription. Other regulatory sequences, such as promoterproximalelements and enhancers, also affect the frequency of transcription.4. In bacteria, a single RNA polymerase produces the primary transcript precursors for allthree major classes of RNA: messenger RNA (mRNA), ribosomal RNA (rRNA), andtransfer RNA (tRNA). Because bacteria do not contain nuclei, ribosomes bind to mRNAas it is being transcribed, and protein synthesis occurs simultaneously with transcription.Eukaryotic genes are transcribed in the nucleus by three different RNA polymerases,each principally responsible for one of the major classes of RNA. The primary transcriptsare modified and trimmed to produce the mature RNAs. The precursors of mRNA(called pre-mRNA) have a guanosine “cap” added at the 5_-end and a poly(A) “tail” atthe 3_-end. Exons, which contain the coding sequences for the proteins, are separatedin pre-mRNA by introns, regions that have no coding function. During splicingreactions, introns are removed and the exons connected to form the mature mRNA. Ineukaryotes, tRNA and rRNA precursors are also modified and trimmed, although not asextensively as pre-mRNA.Nice to know:Clinical case:Anne Niemick is a 4-year-old girl of Mediterranean ancestry whose height and body weight are belowthe 20th percentile for girls of her age.She is listless, tires easily, and complains of loss of appetite and shortnessof breath on exertion. A dullpain has been present in her right upper quadrant for the last 3 months. Her complexion is slate-grayand she appears pale. Initial laboratory studies indicate a severe anemia (decreased red blood cellcount) with a hemoglobin of 6.2 g/dL (reference range, 12–16). A battery of additional hematologic testsshows that Anne has + -thalassemia, intermediate type.The thalassemias are a heterogenous group of hereditary anemias that constitute the most commongene disorder in the world, with a carrier rate of almost 7%. The disease was first discovered in countriesaround the Mediterranean Sea and was named for the Greek word “thalassa” meaning “sea”. However, itis also present in areas extending into India and China that are near the equator. The thalassemiasyndromes are caused by mutations that decrease or abolish the synthesis of the or chains in theadult hemoglobin A tetramer. Individual syndromes are named according to the chain whose synthesis isaffected and the severity of the deficiency. Thus, in° thalassemia, the superscript 0 denotes none ofthe chain is present; in + thalassemia, the + denotes a partial reduction in the synthesis of the chain.More than 170 different mutations have been identified that cause thalassemia; most of these interferewith the transcription of -globin mRNA or its processing or translation.2013 6 Professor H.D.El-Yassin