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Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
Gene Expression Overview
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Gene Expression Overview

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Gene Expression Overview

Gene Expression Overview

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  • 1. GENE EXPRESSION OVERVIEW SALWA HASSAN TEAMA
  • 2. Gene Expression Gene expression process by which a genes information is converted into the structures and functions of a cell by a process of producing a biologically functional molecule of either protein or RNA (gene product) is made. Gene expression is assumed to be controlled at various points in the sequence leading to protein synthesis.
  • 3. Gene Expression Every cell of the body (with a few exceptions) contains a full set of chromosomes and identical genes. Only a fraction of these genes is turned on, however, and it is the subset that is “expressed” that confers unique properties to each cell type. The proper expression of a large number of genes is a critical component of normal growth and development and the maintenance of proper health. Disruptions or changes in gene expression are responsible for many diseases.
  • 4. Protein synthesis is the process in which cells build protein from information in DNAin two major steps: Transcription Synthesis of an RNA that is complementary to one of the strands of DNA according to instruction stored along a specific sequence (a gene) of a DNA molecule. Translation Ribosomes read a messenger RNA and make protein according to its instruction.
  • 5. GENE EXPRESSION OVERVIEW
  • 6. Transcription Source: http://biology.unm.edu/ccouncil/Biology_124/Images/transcription.gif
  • 7. Transcription The overall scheme is similar in bacteria and eukaryotes, but there are significant difference; transcription initiation and transcription termination especially with added complexity of the eukaryotic transcription initiation system.
  • 8. Overall architecture of RNAPs from bacteria (Thermus aquaticus (1HQM) Minakhin et al., 2001), archaea (Sulfolobus shibatae (2Y0S) Wojtas et al., 2011) and eukaryotes (Saccharomyces cerevisiae (1Y1V) Kettenberger et al., 2004) Overall architecture of RNAPs from bacteria (Thermus aquaticus (1HQM) Minakhin et al., 2001), archaea (Sulfolobus shibatae (2Y0S) Wojtas et al., 2011) and eukaryotes (Saccharomyces cerevisiae (1Y1V) Kettenberger et al., 2004) http://www.biologie.uni- regensburg.de/Mikrobio/Thomm/Arbeitsgebiete/rnap_en.html Transcription Enzymes
  • 9. Transcription Enzymes Eukaryotic RNAPolymerase Eukaryotic nuclei contain three RNA polymerases. RNA polymerase I is found in the nucleolus; RNA polymerase II &III are located in the nucleoplasm. The three nuclear RNA polymerase have different roles in transcription. Polymerase I makes a large precursor to the major rRNA (28S,18S and 5.8S rRNA in vertebrates). Polymerase II synthesizes hnRNAs, which are precursors to mRNAs. It also make most small nuclear RNAs (snRNAs). Polymerase III makes the precursor to 5SrRNA, the tRNAs and several other small cellular RNAs. Prokaryotes have one type of RNA polymerase for all types of RNA.
  • 10. The key player in the transcription process is RNA polymerase. The E- coli enzyme is composed of a core, which contains the basic transcription machinery, and a - factors which directs the core to transcribe specific gene.
  • 11. Subunit composition of the RNAPs from the three domains of life (modified from doctoral thesis Zeller M. E.) Source: http://www.biologie.uni-regensburg.de/Mikrobio/Thomm/Arbeitsgebiete/rnap_en.html Subunit composition of the RNAPs
  • 12. Transcription has three phases: initiation, elongation, termination. The following is an outline of the three step in bacteria…
  • 13. Prokaryote genes are grouped in operons
  • 14. Transcription  Transcription is a vital control point in the expression of many genes.  RNA polymerase directs transcription. RNA polymerase is the signal that control transcription. RNA polymerase docks at a promoter and continue as the polymerase elongates the RNA chain and ends when the polymerase reaches a terminator and release the finished transcript.
  • 15. Prokaryotic Transcription Initiation Represented as four steps:  Formation of a closed promoter complex.  Conversion of the closed promoter to an open promoter complex.  Polymerizing the first few nucleotides (up o 10) while the polymerase remain at the promoter.  Promoter clearance
  • 16.  The - factor allows initiation of transcription by causing the RNA polymerase holoenzyme to bind tightly to a promoter. This tight binding depends on local melting of the DNA to form an open promoter complex and is stimulated by . The - factor can therefore select which genes will be transcribed.  Bacterial promoters contain two regions centered at -10 and –35 bp upstream of the transcription start site. In E-coli, there is bear a greater or lesser resemblance to two consensus sequences: TATAAT and TTGACA, respectively.
  • 17. Initiation  The RNA polymerase binding causes the unwinding of the DNA double helix which expose at least 12 bases on the template.  This is followed by initiation of RNA synthesis at this starting point. The transcription bubble moves with the polymerase, exposing the template strand so it can be transcribed.  The RNA polymerase starts building the RNA chain, it assembles ribonucleotides triphosphates: ATP; GTP; CTP and UTP into a strand of RNA.  After the first nucleotide is in place, the polymerase joins a second nucleotide to the first, forming the initial phosphodiester bond in the RNA chain.  After has participated in initiation, it appears to dissociate from the core polymerase to carry out elongation. can be reused by different core polymerase.
  • 18. Elongation  The core continues to elongate the RNA, adding one nucleotide after another to the growing RNA chain.  RNA polymerase directs the sequential binding of riboncleotides to the growing RNA chain in the 5'-3' direction. RNA polymerase moves along DNA template, and the bubble of melted DNA moves with it. This transcription bubble is 10-18 bases long and contain an RNA- DNA hybrid about 9bp long.  Each ribonucleotide is inserted into the growing RNA strand following the rules of base pairing. This process is repeated till the desired RNA length is synthesized……………………..
  • 19. Termination Some regions on the DNA that signal termination (terminators) are recognized by RNA polymerase. Two kinds of terminator:  Intrinsic terminators, function with the RNA polymerase by itself without help of other proteins.  Rho dependent termination. The result is that the RNA transcript dissociate from RNA polymerase and DNA and so stop transcription.
  • 20. Source: http://www.personal.psu.edu/rch8/workmg/TxnRNAPolCh10.htm
  • 21. Eukaryotic Transcription Initiation
  • 22. The general transcription factors combine with RNA polymerase form a preinitiation complex that is competent to initiate transcription as soon as nucleotide are available.
  • 23. Preinitiation Complex First, an RNA polymerase along with general transcription factors binds to the promoter region of the gene to form a closed complex called the preinitiation complex. Preinitiation complex contains:  Core Promoter Sequence  Transcription Factors  RNA Polymerase  Activators and Repressors.
  • 24. Transcription starts upstream from the first coding sequence at the transcription initiation site. RNA polymerase recognizes a promoter. Eukaryotic Promoter lies upstream of the gene. There are several different types of promoter found in human genome, with different structure and different regulatory properties class/I/II/III. One important promoter sequence is the TATA box, a conserved region rich in adenines and thymines, approximately 20-30 bp upstream of the start site of transcription. The TATA box appears to be important for determining the position of the start of transcription. The assembly of the preinitiation complex on each kind of eukaryotic promoter e.g. (class II promoters recognized by RNA polymerase II) begins with the binding of an assembly factor to the promoter. With TATA containing class II and presumably class III) promoters, this factor is TBP, but other promoters have their own assembly factors. This tight binding involves the formation of an open promoter complexes in which the DNA at the transcription start site has melted to allow the polymerase to read it. Eukaryotic Transcription Initiation
  • 25. Source: www.ucl.ac.uk
  • 26. TranscriptionFactors for RNApolymerase II (human cells) Factor No. of subunits Molecular mass (kDa) Functions Functions to Recruit: TFIID: TBP 1 38 Recognize core promoter (TATA) TFIIB TFIID: TAFs 12 15-250 Recognize core promoter (non-TATA); Positive and negative regulation RNA Pol II? TFIIA 2 12, 19, 35 Stabilize TBP-DNA binding; Anti-repression TFIIB 1 35 Select start site for RNA Pol II RNA PolII-TFIIF RNA Pol II 12 10-220 Catalyze RNA synthesis TFIIE TFIIF 2 30, 74 Target RNA PolII to promoter; destabilize non-specific interactions between PolII and DNA TFIIE 2 34, 57 Modulate TFIIH helicase, ATPase and kinase activities; Directly enhance promoter melting? TFIIH TFIIH 9 35-89 Helicase to melt promoter; CTD kinase; promoter clearance? Source: Roeder, R.G. (1996) TIBS 21: 327-335
  • 27. Transcription Factors The class II preinitiation complex contains polymerase II and six transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH. The class general transcription factors and RNA polymerase bind in specific order to the growing preintiation complex (at least in vitro).
  • 28. Mediators,; another collection of proteins and can be considered as a general transcription factor, because it is a part of most, if not all class II preintiation complexes. *Model of RNA Polymerase II Transcription Initiation Machinery. The machinery depicted here encompasses over 85 polypeptides in ten (sub) complexes: core RNA polymerase II (RNAPII) consists of 12 subunits; TFIIH, 9 subunits; TFIIE, 2 subunits; TFIIF, 3 subunits; TFIIB, 1 subunit, TFIID, 14 subunits; core SRB/mediator, more than 16 subunits; Swi/Snf complex, 11 subunits; Srb10 kinase complex, 4 http://www.bio.davidson.edu/courses/genomics/2002/james/favoriteyeastproteins.htmsubunits; and SAGA, 13 subunits. This figure provided by Comprehensive Yeast Genome Database. Source: https://labs.fhcrc.org/hahn/Research/polii_res.html *
  • 29.  The activity of many promoters are greatly increased by sequence called enhancers which can exert their stimulatory actions over distances of several thousands base pairs. Enhancers can be upstream, downstream or even in the midst of transcribed gene.  Activators (gene specific transcription factors) can provide extraboost in transcription. Activators can bind to enhancers and also permits cells to control expression of their genes.
  • 30. Source: http://scienceblogs.com/pharyngula/2007/01/16/basics-what-is-a-gene/
  • 31.  The region downstream of the polyadenylation site is essential for termination.  Cleavage of the nascent transcript at multiple sites downstream of the polyadenylation sites downstream of polyadenylation site is required for termination.  The transcript cleavage occurs cotranscriptionally and presumably preceded cleavage at the polyadenylation site.  The product is immature mRNA Pre mRNA (Primary transcript).  The primary product of RNA transcription; the hnRNAs contain both intronic and exonic sequences.  These hnRNAs are processed in the nucleus to give mature mRNAs that are transported to the cytoplasm where to participate in protein synthesis. Eukaryotic Transcription Termination and mRNA Splicing
  • 32. RNA Processing Capping The cap structure is added to the 5' of the newly transcribed mRNA precursor in the nucleus prior to processing and subsequent transport of the mRNA molecule to the cytoplasm. The 5' cap is a 7-methylguanosine triphosphate. Splicing Step by step removal of introns and joining of remaining exons; it takes place on a special structure called spliceosomes. Addition of poly A tail Synthesis of the poly (A) tail involves cleavage of its 3' end and then the addition of about 40- 200 adenine residues to form a poly (A) tail. Poly A tail appears to increase stability of the resulting polyadenylated RNA.
  • 33. RNA Processing
  • 34. Alternative Splicing Alternative splicing: is a very common phenomenon in higher eukaryotes. It is a way to get more than one protein product out of the same gene and a way to control gene expression in cells.
  • 35. Translation
  • 36. Translation The overall scheme is similar in bacteria and eukaryotes, but there are significant difference, especially added complexity of the eukaryotic translation initiation system. Translation is the process by which ribosomes read the genetic message in the mRNA and produce a protein according to message instruction.
  • 37. The Genetic Code  The purine and pyrimidine bases of the DNA molecule are the letters or alphabet of the genetic code.  Series of codons in part of a mRNA molecule. Each codon consists of three nucleotides.  64 different combination of bases; 61 of them code for 20 amino acid (AA); the last 3 codon (UAG,UGA,UAA) do not code for amino acids, they are termination codons.  The sequence of codons in the mRNA defines the primary structure of the final protein.  Degenerate, specific, no gaps, non overlapping, almost universal,…
  • 38. Requirement for Translation  Ribosomes   tRNA   mRNA template   Amino Acids   Initiation factors   Elongation factors   Termination factors   Aminoacyl tRNA synthetase   Energy source
  • 39. Ribosomes  Factory for protein synthesis.  Composed of ribosomal RNA and ribosomal proteins; known as a Ribonucleoprotein (RNP).  Translate messenger RNA (mRNA) to build polypeptide chains using amino acids delivered by transfer RNA (tRNA).
  • 40. Ribosome Genetic Code
  • 41. Large Ribosomal Subunit Three Sites  A site bind to an aminoacyl tRNA (tRNA bound to an amino acid).  P site bind a peptidyl tRNA (a tRNA bound to peptide being synthesized).  E site binds a free tRNA before it is exist the ribosome.
  • 42. Preparatory Steps for Protein Synthesis First, aminoacyl tRNA synthetase join amino acid to their specific tRNA begin with the activation of amino acids with AMP derived from ATP. Second, ribosomes must dissociate into subunits at the end of each round of translation. The protein synthesis occur in 3 phases: Accurate and efficient initiation occurs, the ribosomes binds to the mRNA, and the first amino acid attached to its tRNA. Chain elongation, the ribosomes adds one amino acid at a time to the growing polypeptide chain Accurate and efficient termination, the ribosomes releases the mRNA and the polypeptide.
  • 43. Translation Initiation  The initiation phase of protein synthesis requires many Initiation Factors.  The small subunit of the ribosome binds to a site "upstream" of the start of the message.  The small subunit of the ribosome proceeds downstream (5' - 3') until it encounters the start codon AUG.  Then the small subunit of the ribosome is joined by the large subunit and a special initiator tRNA.  The initiator tRNA binds to the P site on the ribosome.  In eukaryotes, initiator tRNA carries methionine (Met). Bacteria use (fMet.)
  • 44. Translation (Initiation in Bacteria)  The initiation codon in prokaryotes is usually AUG, but it can also be GUG, or more rarely, UUG.  Dissociation of the 70s ribosomes into 50s and 30s subunits under the influence of IF1  Binding of IF3 to the 30S subunit, which prevents reassociation between the ribosomal subunits.  Binding of IF2,IF2 and GTP alongside IF3.  Binding of mRNA and fMet-tRNAfMet to form the 30S initiation complex. These two components can apparently bind in either order, but IF2 sponsors fMet-tRNAfMet binding, and IF3 sponsors mRNA binding. In each case, the other initiation factors also help.  Binding of the 50S subunit, with loss of IFI and IF3  Dissociation of IF2 from the complex, with simultaneous hydrolysis of GTP. The product is 70 S complex ready to begin elongation.
  • 45. Binding between the 30S prokaryotic ribosomal subunit and the initiation site of an mRNA depends on base pairing between a short RNA sequence called the Shine-Dalgarno sequence just upstream of the initiation codon, and a complementary sequence at the 3' - end of the 16S rRNA. This binding is mediated by IF3, with help from IF1 and IF2. All three initiation factors have bound to the 30S subunit by this time.
  • 46. Eukaryotic Initiation  Eukaryotic 40S ribosomal subunits, together with the initiator tRNA (tRNAiMet), generally locate the appropriate start codon by binding to 5'-cap of an mRNA and scanning downstream until they find the first AUG in a favorable context.  In 5-10% of the cases, the ribosomal subunits will bypass the first AUG and continue to scan for a more favorable one.
  • 47. Eukaryotic Initiation Factors Eukaryotic Initiation Factors, more……….
  • 48. Elongation  The elongation processes in bacteria and eukaryotes are very similar  To begin elongation, another amino acid needed to join the first. The second amino acid arrives bound to tRNA and the nature of this aminoacyl-tRNA is dictated by the second codon in the message. The second codon is in the A site, which otherwise empty. This step requires a protein elongation factor known as EF-TU and GTP in bacteria.  Peptide bond formation: An enzyme peptidyl transferase forms a peptide bond between peptide in the P site and the newly arrived aminoacyl tRNA in the A site. The whole assembly in the A site is dipeptidyl tRNA, and deacylated tRNA remains in the P site (tRNA without its amino acids).  Translocation: the mRNA with its peptidyl tRNA attached in the A site moves one codon's length to the left lead to; the deacylated tRNA in the P sites leaves the ribosomes via the E sites, the dipeptidyl tRNA in the A site, along with its corresponding codon moves into the P site. Translocation requires an elongation factor called EF-G in bacteria ; EF-2 in eukaryotes plus GTP.  The process repeats itself to add another amino acids, and continuous over and over until the ribosomes reaches the last codon in the message. When the polypeptide complete , it is a time for chain termination.
  • 49. Termination  Translational termination requires specific protein factors identified as releasing factors, RFs in E. coli and eRFs in eukaryotes.  The signals for termination are the same in both prokaryotes and eukaryotes. These signals are termination codons present in the mRNA. There are 3 termination codons, UAG, UAA and UGA.  Prokaryotic translation termination is mediated by three factors: RF1,RF2 and RF3. RF1 recognizes the termination codon UAA and UAG; RF2 recognizes UAA and UGA. RF3 is a GTP binding protein that facilitate binding of RF1 and RF2 to the ribosome.  Eukaryotes have two release factors: eRF1 which recognizes all three termination codons, and eRF3, a ribosome dependent GTPase that helps eRF1 release the finished polypeptide.  Ribosomes do not release from mRNA spontaneously after termination, they need help from ribosome recycling factor (RRF) and EF-G in bacteria.
  • 50. ELONGATION
  • 51. Reading the instruction means translating the code in the mRNA from bases building block of DNA to amino acids.
  • 52. Eukaryotic Gene Expression
  • 53. http://employees.csbsju.edu/hjakubowski/classes/ch331/bind/olbindtransciption.html
  • 54. Control of Gene Expression in Eukaryotes
  • 55. Control of Gene Expression in Eukaryotes prevent transcription, prevent mRNA from being synthesized.,Transcriptional control mRNA after it has been produced.,Posttranscriptional prevent translation; involve protein factors needed for translation.,Translational after the protein has been producedPosttranslational,
  • 56. Glossary their sequence of DNA bases.differences inare forms of the same gene with smallAlleles noncodingDNA and/ora segment of a gene that is represented in the mature RNA product. Individual exons may contain codingn:Exo DNA (untranslated sequences). intervening sequence) (A noncoding DNA sequence ): Intervening stretches of DNA that separate exons.(Introns The initial production of gene transcription in the nucleus; an RNA containing copies of all exons and introns.:Primary transcript : RNA molecule that is not translated into a protein. Noncoding RNA genes produce transcriptscoding RNA gene-RNA gene or non that exert their function without ever producing proteins. Non-coding RNA genes include transfer RNA (tRNA) and ribosomal RNA .ncRNAsand lastly longpiRNAssiRNAsand, microRNAs,snoRNAs), small RNAs such asrRNA( are DNA elements that stimulate or depress the transcription of associated genes; they rely on tissue specific:Enhancers and silencers binding proteins for their activities; sometimes a DNA elements can act either as an enhancer or silencer depending on what is bound to it. Activators: Additional gene-specific transcription factors that can bind to enhancer and help in transcription activation. (ORF): A reading frame that is uninterrupted by translation stop codon (reading frame that contains a start codonOpen reading frame and the subsequent translated region, but no stop codon). Directionality: in molecular biology, refers to the end-to-end chemical orientation of a single strand of nucleic acid. The chemical convention of naming carbon atoms in the nucleotide sugar-ring numerically gives rise to a 5' end and a 3' end ( "five prime end" and "three prime end"). The relative positions of structures along a strand of nucleic acid, including genes, transcription factors, and polymerases are usually noted as being either upstream (towards the 5' end) or downstream (towards the 3' end). RNA polymerase, DNA and newly formed RNA.containg: regionTranscription bubble More:http://www.ncbi.nlm.nih.gov/books/NBK7584/
  • 57. References and Further Reading  Ali Khalifa. Applied molecular biology; eds: ( Fathi Tash and Sanna Eissa). 109 pages. Egypt. University Book Center. 2002. Available in paper copy from the publisher  Daniel H. Farkas. DNA Simplified: The Hitchhiker's Guide to DNA. 110 pages. Washington, DC: AACC Press, 1996, ISBN 0-915274-84-1. Available in paper copy from the publisher  Daniel P. Stites, Abba T. Terr. Basic Human Immunology: 336 Pages. Appleton & Lange (November 1990). ISBN. 0838505430. Available in paper copy from the publisher  Innis, David H. Gelfand, John J. Sninsky. PCR Applications: Protocols for Functional Genomics: 566 pages. Academic Press; 1 edition (May 17, 1999). ISBN:0123721865. Available in paper copy from the publisher  Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter. Molecular Biology of the cell. 1392 pages.Garland Science; 5 edition (November 16, 2007).ISBN. 9780815341055. Available in paper copy from the publisher  Robert F. Mueller, Ian D. Young. Emery's Elements of Medical Genetics: Publisher: Churchill Livingstone. 1995 ISBN. 044307125X. Available in paper copy from the publisher.  Robert F. Weaver. Molecular Biology. 600 Pages. Fourth Edition. McGraw-Hill International Edition. ISBN 978-0-07-110216-2.  William B. Coleman, Gregory J. Tsongalis. Molecular Diagnostics. For the Clinical Laboratorian: 592 pages. Humana Press; 4th Printing. edition (August 15, 2005). ISBN 1588293564... Available in paper copy from the publisher.  Eukaryotic promoter . Internet. Available from; http://www.patentlens.net/daisy/promoters/242/g1/250/1346.html Transcription factor. Available from. Fred Hutchinson Cancer Research Center  Transcription factor . Internet. Table. Available from; http://oregonstate.edu/instruction/bb492/lectures/EuTranscriptionI.htm
  • 58. Thank you

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