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Molecular Biology I Lecture 3 - 4: Transcription Prepared By: Osama Hussein...

Molecular Biology I Lecture 3 - 4: Transcription Prepared By: Osama Hussein Presented By: Mahdi Hamed Al-Neelain University, Faculty of Science and Technology, Department of Microbiology and Molecular Biology  Definitions Transcription is the synthesis of a ssRNA copy of a segment of DNA. In the case of protein synthesis, a protein-coding gene is transcribed to give a messenger RNA Translation (protein synthesis) is the conversion of the mRNA base- sequence information into the amino acid sequence of a polypeptide. Osama Hussein 2  Gene Expression—The Central Dogma: An Overview In 1956, Crick gave the name central dogma to the two-step process denoted DNA → RNA → protein (transcription followed by translation). In transcription; only one of the two DNA strands is transcribed into an RNA. Osama Hussein 3 Steps Leading From Gene to Protein in Bacteria and Eucaryotes Osama Hussein 4  There are four main types of RNA molecules, each encoded by its own type of gene, but only one of them is translated: 1. mRNA encodes the amino acid sequence of a polypeptide. mRNAs are the transcripts of protein-coding genes. Translation of an mRNA produces a polypeptide. 2. rRNA (ribosomal RNA), with ribosomal proteins, makes up the ribosomes—the structures on which mRNA is translated. 3. tRNA (transfer RNA) brings amino acids to ribosomes during translation. 4. snRNA (small nuclear RNA), with proteins, forms complexes that are used in eukaryotic RNA processing to produce functional mRNAs. Osama Hussein 5  The Transcription Process Gene regulatory elements: sequence associated with each gene, which are involved in regulating transcription. The enzyme RNA Pol catalyzes the process of transcription The DNA double helix unwinds for a short region next to the gene before transcription begins. In bacteria, RNA Pol is responsible for unwinding; In eukaryotes, unwinding is done by other proteins that bind to the DNA near the start point for transcription. Osama Hussein 6  RNA is synthesized in the 5’-to- 3’ direction. The 3’-to-5’ DNA strand that is read to make the RNA strand is called the template strand. The 5’-to3’ DNA strand complementary to the template strand, and having the same polarity as the resulting RNA strand, is called the nontemplate strand. Osama Hussein 7  DNA vs. RNA Polymerase First, RNA Pol catalyzes the linkage of ribonucleoside triphosphates ATP, GTP, CTP, and UTP, collectively called NTPs nucleoside ( triphosphates) not deoxyribonucleotides. Second, unlike the DNA Pols involved in DNA replication, RNA Pols can start an RNA chain without a primer. RNA Pols make about one mistake for every 104 nucleotides copied into RNA (one every 107 nucleotides by DNA Pol). Osama Hussein 8  In RNA the base U is used instead of T For example, if the template DNA strand reads 3’-ATACTGGAC-5’ then the RNA chain will be synthesized in the 5’-to3’ direction and will have the sequence 5’-UAUGACCUG-3’ Osama Hussein 9 Transcription in Eukaryotes Osama Hussein 10  Eukaryotic RNA Pols Three different RNA Pols transcribe the genes for four main types of RNAs. RNA Pol I, located in the nucleolus, catalyzes the synthesis of three of the RNAs found in ribosomes: - the 28S, 18S, and 5.8S rRNA molecules. RNA Pol II, located in the nucleoplasm, synthesizes - mRNAs and some snRNAs. RNA Pol III, located in the nucleoplasm, synthesizes: - tRNAs; 5S rRNA, a small rRNA molecule found in each ribosome; and the snRNAs not made by RNA Pol II. Osama Hussein 11  Promoters and Enhancers Promoters of protein-coding genes encompass about 200 base pairs upstream of the transcription initiation site and contain various sequence elements. Two general regions of the promoter are: - the core promoter (CP); and - promoter-proximal elements (PPEs). Osama Hussein 12  The CP is the set of cis-acting sequence elements needed for the transcription machinery to start RNA synthesis at the correct site. These elements are typically within no more than 50 bp upstream of that site. The best-characterized CP elements are: 1. a short sequence element called Inr (initiator), which spans the transcription initiation start site (defined as +1); has the seven- nucleotide consensus sequence 5’-TATAAAA-3’and 2. the TATA box, or TATA element (also called the Goldberg-Hogness box), located at about position-30. The Inr and TATA elements specify where the transcription machinery assembles and determine where transcription will begin. In the absence of other elements, transcription will occur only at a very low level. Osama Hussein 13  PPEs are upstream from the TATAbox, in the area from-50 to-200 nucleotides from the start site of transcription. Examples of these elements are the CAAT (“cat”) box, named for its consensus sequence and located at about-75; and the GC box, with consensus sequence 5’-GGGCGG-3’, located at about -90. Both the CAAT box and the GC box work in either orientation (meaning with the sequence element oriented either toward or away from the direction of transcription). Osama Hussein 14 RNA polymerase II transcriptional control Osama Hussein 15  Promoters contain various combinations of CPEs and PPEs that together determine promoter function. The PPEs are important in determining how and when a gene is expressed. Key to this regulation are transcription regulatory proteins called activators, which determine the efficiency of transcription initiation. For example: - Housekeeping genes, expressed in all cell types (e.g. actin gene) have PPEs that are recognized by activators found in all cell types. - Genes that are expressed only in particular cell types or at particular times have PPEs recognized by activators in those cell types or at those particular times. Osama Hussein 16  Eenhancers are: - required for the maximal transcription of a gene (cis-acting element). - function either upstream or downstream from the transcription initiation site (commonly upstream of the gene they control). - modulate transcription from a distance. - contain a variety of short sequence elements, some of them the same as those found in the promoter. Activators also bind to these elements and with other protein complexes. The DNA containing the enhancer is brought close to the promoter DNA to which the transcription machinery is bound, stimulating transcription to the maximal level for the particular gene. Osama Hussein 17 Steps in transcription Promoter recognition Preinitiation complex formation Initiation Elongation Termination Osama Hussein 18  Transcription Initiation Assembly of RNA Pol II and a number of other proteins called general transcription factors (GTFs) on the core promoter. GTFs bind first and recruit the RNA Pol II to form a complex. Other GTFs then bind, and transcription can begin. GTFs: - position eucaryotic RNA Pol correctly at the promoter, - aid in pulling apart the two strands of DNA to allow transcription to begin, and - release RNA Pol from the promoter into the elongation mode once transcription has begun. Osama Hussein 19 Osama Hussein 20  Three-Dimensional Structure of TBP (TATA-binding protein) The TBP is the subunit of the general transcription factor TFIID that is responsible for recognizing and binding to the TATA box sequence in the DNA. Osama Hussein 21 Initiation of Transcription of Eucaryotic Gene (A) The promoter contains a DNA sequence called the TATA box, which is located 25 nucleotides away from the site at which transcription is initiated. (B) Through its subunit TBP, TFIID recognizes and binds the TATA box, which then enables the adjacent binding of TFIIB Osama Hussein 22  Transcription Initiation by RNA Polymerase II in Eucaryotic Cell Osama Hussein 23  The Structure and Production of Eukaryotic mRNAs The mature, biologically active mRNA in both prokaryotic and eukaryotic cells has three main parts: 1. A 5’ untranslated region (also called a leader sequence) at the 5’ end; 2. The protein-coding sequence, which specifies the amino acid sequence of a protein during translation; and 3. A 3’ untranslated region (also called a trailer sequence). Osama Hussein 24  mRNA production is different in bacteria and eukaryotes In eukaryotes: - the RNA transcript (the pre-mRNA) is modified in the nucleus by RNA processing to produce the mature mRNA. - the mRNA must migrate from the nucleus to the cytoplasm. - mRNA is always transcribed completely and then processed before it is translated. - eukaryotic mRNAs typically are monocistronic, meaning that they contain the amino acid-coding information from just one gene. Osama Hussein 25 Osama Hussein 26 Production of Mature mRNA in Eukaryotes Osama Hussein 27  5 Modification (5 capping) Once RNA polymerase II has made about 20 to 30 nucleotides of pre-mRNA, a capping enzyme adds a guanine nucleotide—most commonly, 7-methyl guanosine (m7G)—to the 5’ end. The addition involves an unusual linkage. The sugars of the next two nucleotides are also modified by methylation. The 5’ cap remains - Protect the mRNA against degradation by exonucleases. - Important for the binding of the ribosome as an initial step of translation Osama Hussein 28 Co-transcriptional capping Osama Hussein 29  3’ Modification Most eukaryotic pre-mRNAs become modified at their 3’ ends by the addition of a sequence of about 50 to 250 adenine nucleotides called a poly(A) tail. There is no DNA template for the poly(A) tail. The poly(A) tail - Required for efficient export of the mRNA from the nucleus to the cytoplasm. - Protects the 3’ end of the mRNA against early degradation by exonucleases. - Plays important roles in the initiation of translation by ribosomes - Stabilizes mRNA. Osama Hussein 30  Pre-mRNA processing Introns typically begin with 5’-GU and end with AG-3’, although more than just those nucleotides are needed to specify a junction between an intron and an exon. Introns in pre-mRNAs are removed and exons joined in the nucleus by mRNA splicing. The splicing events occur in a spliceosome, a complex of the pre- mRNA bound to small nuclear ribonucleoprotein particles (snRNPs; pronounced snurps). snRNPs are small nuclear RNAs (snRNAs) associated with proteins. The five principal snRNAs are U1, U2, U4, U5, and U6; each is associated with a number of proteins to form the snRNPs. Osama Hussein 31  intron-3′ exon lariat 5′ exon Osama Hussein 32 Osama Hussein 33