1.Dr. Faisal Al-AllafAssistant Professor of Geneticsand Molecular MedicineUmm Al-Qura University Faculty ofMedicine, Makkah, Saudi Arabiafallaf@uqu.edu.saTel/Fax: 5270000 Ext: 4198The Cellular andMolecular Basisof Inheritance1431/04/11 Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 1

2. . . . . :http://el.uqu.edu.sa/jusur/index.php?un_id=uqu :http://www.uqu.edu.sa/faallaf 11/40/1341Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa2 3. Course contents (syllabus)GENOME, TRANSCRIPTOME, AND PROTEOMECell, DNA and RNAGene structure and genetic codesCell cycle and DNA replicationTranscription and post-transcriptional modificationRNA and regulation of gene expressionTranslation and post-translational modificationCHROMOSOMES AND CELL DIVISIONChromosomes morphology and classificationCell cycle divisionMitosisMeiosis1431/04/11 Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 3 4. Comparison between DNA and RNAThere are two types of Nucleic acids. DNA exists as twostrands, twisted together into a right handed helix, calledthe double helix. Each strand is a polymer of repeating unitscalled nucleotides.Feature DNARNADeoxyribose at the position 2Sugar of the ribose sugar RiboseBase pairingA-T and G-CA-U and G-C Single strandedStructure Double helix structureStrand length Long Short Carries protein-encodingMaintain RNA-, and information andprotein-encoding controls howFunctioninformation. information is used.DNA cannot function as RNA can function as anan enzyme enzyme1431/04/11Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 4 5. Types of RNAIn eukaryotic cells, all RNA is produced from DNA bytranscriptionRNA is synthesized predominantly in the nucleus, butmoves out into the cytoplasm to carry out its functionEukaryotic RNAs must be transported from the nucleusto the cytoplasm through the nuclear pore complexesThere are three main types of RNAMessenger RNA (mRNA)Ribosomal RNA (rRNA)Transfer RNA (tRNA)Other types of RNA include: heteronuclear RNA(hnRNA), small nuclear RNA (snRNA), small nucleolarRNA (snoRNA), microRNA, and mitochondrial RNA1431/04/11 Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 5 6. Transfer RNA (tRNA)It transports or carries specific amino acids to theribosome for protein synthesis. So it acts as anadaptors to correctly order amino acids onmRNA for protein synthesis.tRNA is a small linear molecule with 73 93nucleotides (an average of 76 nucleotides) andlower MW than rRNA.Transfer RNA is unusual containing a variety ofrare bases in addition to C, G, A, and U some ofthese are modified by methylation.There are over 40 different tRNA subfamilies,each with several member.1431/04/11Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 6 7. Transfer RNA (tRNA)Exhibits extensive intra molecular basepairing which could adopt varioussecondary structures. The most notable isa cloverleaf shaped secondary structurewhich then twists into an L shapestructure.The clover leaf shape consists of fivearmsThe clover leaf shape has two active sites,which allow it to carry out its function.1431/04/11Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 7 8. Transfer RNA (tRNA)The first active site is on the acceptorarm, which is formed by seven basepairs between the 5 and the 3 ends ofthe molecules. At the 3 terminal CCAgroup can accept (bind) a specificamino acidThe second active site is at theanticodon arm. It contains the triplet ofnucleotides called the anticodon whichbase pair with the mRNA duringtranslation (It is recognises or bind thecorresponding (complementary) mRNAcodon).1431/04/11 Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 8 9. Ribosomal RNA (rRNA)Ribosomal RNA is a component of ribosomes. It is a large moleculethat joins with (55) various proteins to form ribosome, whichstructurally support and catalyze protein synthesis.It is vital in translation. Its function is correct orientation ofamino acids.In eukaryotic cell, each ribosomeconsists of two unequal subunites, onesmall called the S (small) and one largecalled the L (large) subunits.Each ribosome contain many proteinsderived by translation of mRNA, plusribosomesRNA that remain untranslatedElectron micrograph of Endoplasmic ReticulumIt undergoes extensive intra-molecular base pairing which isimportant in determining the ribosomal structure.1431/04/11 Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa9 10. Heteronuclear RNA (hnRNA) Heteronuclear RNA is the primary transcript produced by eukaryotic cells. Unlike the final mRNA transcript, it contains introns that are removed by RNA splicing. The conversion of hnRNA into mRNA by the removal of introns occurs in the nucleus in RNA-protein complexes called splicesomes.1431/04/11Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 10 11. Messenger RNA (mRNA)Messenger RNA encodes amino acid sequenceMessenger RNA carries genetic information from the nucleus into thecytoplasm. It is the transcript of a protein coding gene.In eukaryotes it is derived by splicing the initial RNA transcript (hnRNA).It forms the templates upon which polypeptides are manufactured duringtranslation.It is a single stranded molecules without any intramolecular hydrogenbonds.1431/04/11Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa11 12. Transcription and RNA processingTranscription is the process wherebygenetic information is transmitted fromDNA to RNA or mRNA. Thus, mRNAis synthesized according to the DNAtemplate.The process is catalyzed by DNA-dependent RNA polymerases, whichrecognize different promoters andtherefore, transcribe different types ofRNA molecules.Transcription occursinthreesequential phases:InitiationElongationTermination1431/04/11 Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 12 13. Transcription and RNAsynthesisThe DNA template displays polarity in that only one strand can act asa template (template strand). The non-transcribed strand is called thecoding strand as it has the same base composition as the RNA exceptthat thymines (Ts) are substituted for uracils (Us).The template strand is transcribed. It is indentified by RNA sequencesthat comprise the promoter. Transcription of a gene may beinfluenced and regulated by both cis and trans acting factors:Cis acting factors are specific sequences of DNA that lie the samemolecule of DNA as the gene they regulate.Trans acting factors are proteins that bind to cis acting elements.They are transcribed from genes distinct from the ones theyregulate.1431/04/11Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 13 14. Transcription initiation Transcription is signalled by assembly of protein transcription factors at the promoter. Transcription factors and RNA polymerase are attached to a promoter (a special sequence that signals the start of the gene and indicates where RNA polymerase should begin its action). The promoters for genes that code for a luxury proteins include a TATA box with a sequence that is a variant of 5-TATAAA-3 at about 25 bp upstream of the transcription initiation site. Genes that code for housekeeping proteins instead usually have one or more GC boxes in variable positions, containing a variant of 5-GGGCGG-3. Another common promoter element is the CAAT box. Transcription factors are proteins that bind to promoter sequences and initiate transcription. Typically they contain an active domain and a DNA binding domain. The DNA binding domains are of four types: the leucin zipper, the helix-loop-helix, the helix turn helix and the zinc finger.1431/04/11 Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa14 15. Transcription initiation Extracellular signals alter the chromatin structure and exposes the promoter of a gene whose transcription is required under the particular conditions. Physiological levels of transcription require the presence of enhancers. Enhancers are cis acting elements that may be located upstream, downstream, intra-gene, and may be many kilobases away from the start of transcription in a 5 or 3 direction. Enhancers are bound by trans acting trans-activating protein, which are then able to associate with the polymerase complex to increase the level of transcription. Introns begins with the sequence GT and end with a run of Cs or Ts preceding AG.1431/04/11 Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa15 16. Transcription elongationRNA polymerase unwind the dsDNA double helixlocally to expose unpaired bases upon which DNA-RNA hybrids form. RNA nucleotides bond withexposed complementary bases on the DNAtemplate strand.Transcription complex including the polymerasethen moves downstream, rather like the sliderdown a zip causing local unwinding and splittingfollowed by reformation of the double helix as itproceeds, creating a 17bp long transcriptionbubble.RNA polymerase adds the RNA nucleotides in thesequence the DNA specifies, moving along theDNA strand in a 3 to 5 direction. Synthesizing theRNA molecule in a 5 to 3 direction.Transcription rate in humans is 20 bases persecond.1431/04/11 Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 16 17. Transcription elongationIn any particular gene only one DNA strand ofthe double helix acts as template strand. Thecoding strand is identical to that of the RNAexcept with the T in place of the USince mRNA is relatively short-lived, a cell mustconstantly transcribe certain genes to maintainsupplies of essential proteins.Different genes on the same chromosome maybe transcribed from different strands of thedouble helix.If the DNA template strand has a sequence of CCTCTACThe transcript will be GGAGAUGThe coding DNA isGGAGATG1431/04/11 Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 17 18. Transcription terminationA terminator sequence in theDNA indicates where the genesRNA-encoding region ends.1431/04/11 Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 18 19. RNA processing Before the primary RNA molecule leaves the nucleus, it undergoes a number of modifications or what is known aspost-transcriptional processing. Post-transcriptional processing involved: 5 capping Polyadenylation Introns excision and exons splicing (RNA splicing)1431/04/11 Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 19 20. 5 cappingAs hnRNA transcripts are synthesized, they are covalently modified tomark them as coded messages for later translation into polypeptides.The 5 end of the primary RNA (hnRNA) is first capped by addition of7-methyl GTP (methylated guanine nucleotide) in reverse orientation.Both the 5 cap and the poly A tail are thought to Facilitate export of the mRNA to the cytoplasm Provide recognition signals to the attachment to the ribosomes Protect the RNA transcript from degradation by endogenous cellular exonucleases. It aids in splicing (the removal of introns from hnRNA)1431/04/11Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 20 21. 3 cleavage at the AAUAAA sequenceand addition of a poly(A) tail When the polyadenylation site appears in the hnRNA strand during transcription, the transcript is cleaved by enzymes 10-20 nucleotides downstream from the polyadenylation signal (AAUAAA). The cleavage gives the 3 end of the transcript a well defined end. The enzyme poly A polymerase adds on 100-200bp residues of adenylic acid to form the poly A tail. The substrate is adenosine triphosphate (ATP). Both the 5 cap and the poly A tail are thought to Facilitates export of the mRNA to the cytoplasm Provides recognition signals to the attachment to the ribosomes Protect the RNA transcript from degradation by endogenous cellular exonucleases.1431/04/11 Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 21 22. RNA splicingAfter transcription, the non-coding introns in the primary RNA (hnRNA) areremoved (introns excisions), and the non-adjacent coding exons are splicedtogether (exons splicing) to form a shorter mature mRNA.The ribonucleoprotein complexes that removes the introns are calledspliceosomes and they contain several snRNA species (U1-U6).The splicing process depends on the existence of consensus sequenceswithin the hnRNA intron, which are recognized by components of thespliceosome: The first two nucleotides of the intron are always GU, which binds U1 and form the splice donor site. An A nucleotide approximately 30 nucleotides from the 3 end of the intron binds U2 and forms the branch point. The last two nucleotides of the intron are always AG, which binds U5 and forms the splice acceptor site.1431/04/11 Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 22 23. RNA splicing The binding of the spliceosome to the intron causes it to loop. The splicing reaction then proceeds in two stages: 1. The first stage is releasing the first exon 2. The second stage is releasing the next exon(s) After the reaction is complete, the exons are joined together and the intron sequence is released as a lariat1431/04/11Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 23 24. Transcription and RNAprocessing1431/04/11 Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 24 25. 1431/04/11 Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 25 26. 1431/04/11 Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 26 27. References and Private Reading These slides are only a handout and the students must read the text book (Emeryselement of medical genetics)1. Emerys Elements of Medical Genetics, 13th edition 2007, by Peter TURNPENNY and Sian ELLARD. Churchill Livingstone ELSEVIER. ISBN: 978-0-7020-2917-22. Medical Genetics at a Glance, 2nd edition 2008, by Dorian PRITCHARD and Bruce KORF. Blackwell Publishing. ISBN: 978-1-4051-4846-73. Genetics for Dummies, 2005, by Tara Robinson, Wiley Publishing, Inc. ISBN: 978- 0-7645-9554-74. Cell Biology and Genetics, Crash Course, 2nd edition 2006, by Manson, Jones, Morris, Michael STEEL and Dan HORTON-SZAR. MOSBY ELSEVIER. ISBN: 0- 7234-3248-15. Human Molecular Genetics, 3rd edition, 2003, by STRACHAN T. and A. READ. Garland science/Taylor and Francis group. ISBN: 978-0-8153-4182-66. Genomes, 3rd edition 2006, by T.A. BROWN. Garland science, ISBN: 978-0-8153- 4138-31431/04/11 Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa27 28. Acknowledgments For the providers of all the educational materials (video clips, pictures, diagrams and charts) including publishers, pharmaceutical companies or unknown internet users who made their material available for use, in this and other presentations, I offer heartfelt thanks and deep appreciation. I feel particularly grateful to faculty, staff, and our brilliant students who provided a unique intellectual and wonderful environment for work.1431/04/11Dr. Faisal Al-Allaf, fallaf@uqu.edu.sa 28