Molecular Biology I BIO316 Lecture 6 PDF

Molecular Biology I BIO316 Lecture 6 Gene Expression: Transcription in Eukaryotes Prepared by Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics ...

Molecular Biology I BIO316 Lecture 6 Gene Expression: Transcription in Eukaryotes Prepared by Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli  Central dogma of Molecular Biology Associate Professor of Biochemistry and Molecular Genetics ➔ In molecular biology, central dogma illustrates the flow of genetic information from DNA to RNA to protein. ⚫ It is defined as a process in which the information in DNA is converted into a functional product.  Difference between Gene and Genome Genes have complex structure Characteristic Gene Genome  Genome vs. Gene: Definition Gene is a segment of DNA that gives The whole nuclear DNA of a cell in an instruction for the synthesis of gene organism comprises its genome.  A gene is the inherited factor that determines a products such as RNA or protein. characteristic. Structure It is the basic unit of heredity. It includes all the coding and non- coding DNA, along with mitochondrial  A genome is the organism's ensemble of genes. and chloroplast DNA. ➔ A gene is a set of nucleotides that specify the amino Length A gene is a few hundred base pairs A genome of an organism has millions long. of base pairs. acids of a protein. Study The study of genes is known as The study of genomes is known as genetics. genomics. Evolution Variations of genes are called alleles. Gene duplication brings out variation in Every gene has two alleles which a genome. contribute to evolution.  The concept of colinearity suggests that the number of nucleotides in a gene should be proportional to the number of amino acids in the protein encoded by that gene.  This concept is true for genes found in bacterial cells.  In eukaryotes, not all genes are colinear with the proteins that they encode. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Difference between Exons and Introns Exon vs. Intron Exons Introns ➔ Many eukaryotic genes contain coding regions called Found in both prokaryotes and eukaryotes Found in Eukaryotes only exons and noncoding regions called interrupting Coding areas of the DNA Non-coding areas of the DNA sequences or introns. Exons are the nucleotide sequence in mRNA, Introns are the non-coding part which are  All the introns and the exons are initially transcribed which codes for proteins removed before translation by RNA splicing Exons are highly conserved The sequence of the introns frequently changes into RNA. over time. They are less conserved  After transcription, the introns are removed by splicing DNA bases that are translated into proteins DNA bases found in between exons and the exons are joined to yield the mature RNA. Mature mRNA contains exons and moves to the Introns are removed in the nucleus before the cytoplasm from the nucleus mRNA moves to the cytoplasm  Introns are common in eukaryotic genes but are rare in bacterial genes.  The number and size of introns vary widely in eukaryotes.  Most eukaryotic genes contain more noncoding nucleotides than coding nucleotides.  Most introns do not encode proteins. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  The Three RNA Polymerases in Eukaryotic Cells Transcription in Eukaryotes Type of polymerase Genes transcribed RNA polymerase I 5.8S, 18S, and 28S rRNA genes ➔ Eukaryotic nuclei have three major enzymes:  RNA polymerase I. RNA polymerase II mRNA genes snRNA, snoRNA, lncRNA, miRNA genes  RNA polymerase II. RNA polymerase III tRNA genes, and 5S rRNA genes  RNA polymerase III. snRNA, miRNA genes  The three polymerases have similar structures, but The rRNAs were named according to their “S” values, which refer to their rate of they transcribe different categories of genes. sedimentation in an ultracentrifuge. The larger the S value, the larger the rRNA.  Eukaryotic RNA polymerase II has many structural similarities to bacterial RNA polymerase.  Eukaryotic RNA polymerases require many factors called the transcription factors.  The initiation of Eukaryotic transcription take place on DNA that is packaged into nucleosomes and higher-order forms of chromatin structure, features that are absent from bacterial chromosomes. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Transcription in Eukaryotes Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics ➔ Transcription is the process by which the information in a DNA strand is copied into a new molecule of RNA.  It is the first step of gene expression, in which a particular segment of DNA is copied into mRNA by the enzyme RNA polymerase.  It results in a complementary, antiparallel mRNA strand called a primary transcript. Enzymes involved in transcription  The nucleus of a eukaryotic cell has three RNA polymerases responsible for transcribing different types of RNA.  RNA polymerase I presents in the nucleolus and transcribes the 5.8S, 18S, and 28S rRNA genes.  RNA polymerase II located in the nucleoplasm and transcribes protein-coding genes, to yield pre-mRNA.  RNA polymerase III located in the nucleoplasm. It transcribes the tRNA, 5S rRNA genes.  Dr. Rami Elshazli Features of Eukaryotic Transcription Associate Professor of Biochemistry and Molecular Genetics ➔ Eukaryotic Transcription occurs within the nucleus and mRNA moves out of the nucleus into the cytoplasm for translation. ➔ The basic mechanism of RNA synthesis by eukaryotic RNA polymerases can be divided into:  Initiation phase ➔ During initiation, RNA polymerase recognizes a specific site on the DNA, upstream from the gene, called a promoter site and then unwinds the DNA.  Most promoter sites for RNA polymerase II include a highly conserved sequence located about 25–35 bp upstream of the start site which has the consensus 5’- TATAAA-3’ and is called the TATA box.  Since the start site is denoted as position +1, the TATA box position is said to be located at position -25 to -35.   Initiation phase Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Some eukaryotic protein-coding genes lack TATA box and have an initiator element instead, centered around the transcriptional initiation site.  To initiate transcription, RNA polymerase II requires the assistance of other proteins complexes, called basal transcription factors.  These factors must assemble into a complex on the promoter for RNA polymerase to bind and start transcription. ➔ These have the generic name of TFII (for Transcription Factor for RNA polymerase II).  The first event in initiation is the binding of the transcription factor IID (TFIID) protein complex to the TATA box via its subunits called TBP (TATA box binding protein).  As soon as the TFIID complex has bound, TFIIB binds and stabilizes the TFIID-TATA box interaction.  Next, TFIIA binds to TFIID.   Initiation phase Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics ➔ The RNA polymerase II and the rest of the general transcription factors assemble at the promoter.  This is followed by the binding of TFIIF, TFIIH and TFIIE.  This final protein complex contains at least 40 polypeptides and is called the transcription initiation complex.  RNA polymerase II contains long C-terminal polypeptide tail that is called the C-terminal domain (CTD).  Dr. Rami Elshazli  Initiation phase Associate Professor of Biochemistry and Molecular Genetics Basal Transcription Factors Needed for initiation phase Name Number of subunits Major functions TFIID 12 - Recognizes TATA box near the transcription start point at the promoter site TFIIB 1 - Positions RNA polymerase II at the start site of transcription TFIIA 2 - Stabilizes binding of TFIID TFIIF 3 - Stabilizes RNA polymerase interaction with TFIIB - Helps attract TFIIE and TFIIH TFIIE 2 - Attracts and regulates TFIIH TFIIH 10 - Unwinds DNA at the transcription start point. - Phosphorylates the RNA polymerase C-terminal domain (CTD). - Releases RNA polymerase from the promoter. TFIID is composed of TBP and 11 additional subunits  Dr. Rami Elshazli  Elongation phase Associate Professor of Biochemistry and Molecular Genetics ➔ TFIIH has two functions:  It is a helicase; it can use ATP hydrolysis to unwind the DNA helix, allowing transcription to begin.  It phosphorylates RNA polymerase II and causes conformational changes in the enzyme to dissociate from other proteins in the initiation complex. ➔ The key phosphorylation occurs on a long C-terminal tail called the C-terminal domain (CTD) of the RNA polymerase II molecule.  Only RNA polymerase II with a phosphorylated CTD can elongate RNA.  RNA polymerase II starts moving along the DNA template, synthesizing RNA, that is, the process enters the elongation phase.  RNA synthesis occurs in the 5’ → 3’ direction.  The RNA molecule made from a protein-coding gene by RNA polymerase II is called a primary transcript.  Dr. Rami Elshazli  Termination phase Associate Professor of Biochemistry and Molecular Genetics ➔ Elongation of the RNA chain continues until termination occurs.  Transcription can stop at varying distances downstream of the gene.  RNA Polymerase II can continue to transcribe RNA from a few bp to thousands of bp. ➔ The transcript is cleaved at an internal site before RNA Polymerase II finishes transcribing.  This releases the upstream portion of the transcript, which will serve as the initial RNA = primary transcript.  This cleavage site has 5′-exonulease activity that stimulate the termination step and the release of mRNA molecule.  Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics