Cell Biology Lecture 7: Transcription PDF

Cell Biology Lecture 7: TRANSCRIPTION From CHAPTER 6 Molecular Biology of the Cell, 4th Ed. Alberts et al. Daniele Provenzano, Ph.D. REVIEW: The pathway of gene expression (the flow of information) begins from DNA, transcribed into the intermediate RN...

Cell Biology Lecture 7: TRANSCRIPTION From CHAPTER 6 Molecular Biology of the Cell, 4th Ed. Alberts et al. Daniele Provenzano, Ph.D. REVIEW: The pathway of gene expression (the flow of information) begins from DNA, transcribed into the intermediate RNA leading to translation of protein, the final product.  REVIEW: Genes can be EXPRESSED with different efficiencies. For example, gene A can give rise to a lot of protein while gene B can yield only a little. Both transcription and translation can mediate the regulation of gene expression. The portions of the gene that are transcribed include the OPEN READING FRAME or CODING sequence plus additional regulatory sequences. NOT ALL DNA IS TRANSCRIBED AND TRANSLATED  REVIEW: RNA: DNA: Similarities between DNA and RNA: (A) The phosphodiester backbone is the same. (B) Much like thymine base pairs with adenine, so does uracil base pair with adenine. (B) (A)  REVIEW: In cells RNA is single-stranded and DNA is double-stranded allowing RNA to take on 3D structures as mentioned before:  REVIEW: TRANSCRIPTION: RNA synthesis from a DNA template TRANSCRIPTION IS MEDIATED BY RNA POLYMERASE Equivalent complementary The RNA molecule synthesized during transcription is complementary to the template DNA strand and thereby equivalent (the same) as the non-coding complementary DNA strand. RNA polymerase: RNA POLYMERASE is a complex enzyme that mediates TRANSCRIPTION. To allow RNA POL to dock onto the DNA the double stranded DNA must be opened up, like in DNA synthesis. However, in transcription this opening of the dsDNA is temporary and performed by RNA POL itself.  RNA POL / DNA POL comparison: 1. RNA POL links RIBONUCLEIC ACIDS not DEOXYRIBONUCLEIC ACIDS. 2. RNA POL does not need an existing primer to begin synthesis of the polymer, it can do this from “scratch”. 3. Because of the temporary nature of the RNA transcript, RNA POL incorporates errors more frequently than DNA POL, 1 every 10’000 nucleotides (104). RNA POL: DNA POL:  RNA POL proofreading: RNA POL also has a proofreading mechanism. When a wrong nucleotide is incorporated, RNA POL backs up and unpolymerizes (undoes) the nucleotides up to the error, a process that is time consuming and inefficient because it removes also correct nucleotides. PROKARYOTIC RNA POL: Unlike in DNA synthesis, the newly formed chain of RNA is detached from the template DNA readily, allowing numerous transcripts (RNA) to be synthesized in sequence, one after the other, at a rate of over a 1000 transcripts per hour. Shown here is an actual electron micrograph image of transcription taking place on two genes: GENE 1: GENE 2: DNA RNA POLYMERASES Freshly synthesized RNA etc… etc…  The Products of RNA POL: The great majority of TRANSCRIPTS generated by RNA POL are called mRNA because they act as templates (messengers) for the synthesis of proteins. A minority of genes encodes sequences that are intended to be RNA as the final product. Not all of the roles of these terminal RNAs are understood, but here are some examples of terminal RNA: 1. Small nuclear RNA (snRNA), these direct splicing of pre-mRNA to the final mRNA product. 2. Ribosomal RNA (rRNA) that compose the core of ribosomes (a hybrid molecule that mediates translation made up in part by protein and in part by RNA), 3. Transfer RNA (tRNA), molecules that form the adaptors that select amino acids and hold them into place on a ribosome for incorporation into proteins. Most of the dry weight of RNA in a cell is rRNA, while mRNA is made transiently and usually has a very short ½ life. The Products of RNA POL: THESE go on to be translated !!! What part of a gene IS transcribed by RNA POL ? TRANSCRIPTION UNITS MADE UP OF: I) THE OPEN READING FRAME, the DNA sequence that encodes for the protein. II) The transcriptional START, where RNA POLYMERASE begins transcription. III) Ribosomal binding site (RBS), where RIBOSOMES will find the transcript to translate in the next step of gene expression. IV) The transcriptional STOP, where RNA POLYMERASE ends transcription. RBS +1 TRANSCRIPTION TRANSLATION What part of a gene IS transcribed by RNA POL ? TRANSCRIPTION UNITS MADE UP OF: I) THE OPEN READING FRAME, the DNA sequence that encodes for the protein. II) The transcriptional START, where RNA POLYMERASE begins transcription. III) Ribosomal binding site (RBS), where RIBOSOMES will find the transcript to translate in the next step of gene expression. IV) The transcriptional STOP, where RNA POLYMERASE ends transcription. RBS +1 TRANSCRIPTION TRANSLATION What part of a gene IS transcribed by RNA POL ? TRANSCRIPTION UNITS MADE UP OF: I) THE OPEN READING FRAME, the DNA sequence that encodes for the protein. II) The transcriptional START, where RNA POLYMERASE begins transcription. III) Ribosomal binding site (RBS), where RIBOSOMES will find the transcript to translate in the next step of gene expression. IV) The transcriptional STOP, where RNA POLYMERASE ends transcription. RBS +1 TRANSCRIPTION TRANSLATION What part of a gene IS transcribed by RNA POL ? TRANSCRIPTION UNITS MADE UP OF: I) THE OPEN READING FRAME, the DNA sequence that encodes for the protein. II) The transcriptional START, where RNA POLYMERASE begins transcription. III) Ribosomal binding site (RBS), where RIBOSOMES will find the transcript to translate in the next step of gene expression. IV) The transcriptional STOP, where RNA POLYMERASE ends transcription. RBS +1 TRANSCRIPTION TRANSLATION What DNA sequence does RNA POL recognize before beginning transcription ? Located ahead of the transcriptional start, the PROMOTER sequence is recognized by RNA POL as the “docking site” before initiating transcription. The promoter sequence is not transcribed, because it is located ahead of the transcriptional start, also known as +1 - the first nucleotide transcribed by RNA POL. promoter RBS +1 TRANSCRIPTION TRANSLATION  RNA POL TRANSCRIPTION TRANSCRIPTION is divided into three events: 1. Transcriptional INITIATION: docking of RNA POL at the promoter promoter ORF terminator 1. ELONGATION: active transcription (the making of the RNA molecule) promoter ORF terminator 1. Transcriptional TERMINATION: the end of transcription, when RNA POL stops promoter ORF terminator Bacterial transcription: https://www.youtube.com/watch?v=dZaSzCgG64s PROKARYOTIC TRANSCRIPTION In bacteria, RNA POL requires accessory proteins, detachable units of RNA POL called SIGMA (σ) FACTORS to recognize promoters. There are several σ FACTORS in bacteria, each recognizes a different group of promoters. σ FACTORS TOGETHER with RNA POL recognize the PROMOTER in the DNA sequence. After transcription begins, the σ FACTOR falls of the RNA POL and ELONGATION proceeds. Transcriptional TERMINATION comes about in two ways; in one, when the mRNA synthesized by RNA POL forms a secondary structure (stem and loop) that “bumps” RNA POL off the RNA transcript. PROKARYOTIC TRANSCRIPTION TERMINATION: Rho-dependent: Rho-independent: Bacterial promoters (and terminators) vary from bacteria to bacteria and even within the same organism, but they do show some patterns: (A) Promoter sequences are located at –10 and –35 from the first nucleotide transcribed (referred to as +1) and consist of two HEXAMERS (two stretches of 6 nucleotides each) whose nucleotides vary but TTGACA-TATAAT is favored. (B) The spacing between the two hexamers can range from 15 to 19 nucleotides but a 17 nucleotide spacer is favored. Promoter sequences, although not identical to one another display “consensus”, a pattern of sequence conservation in which some nucleotides are preferred over others at any given position. Bacterial promoters (and terminators) vary from bacteria to bacteria and even within the same organism, but they do show some patterns: (A) Promoter sequences are located at –10 and –35 from the first nucleotide transcribed (referred to as +1) and consist of two HEXAMERS (two stretches of 6 nucleotides each) whose nucleotides vary but TTGACA-TATAAT is favored. (B) The spacing between the two hexamers can range from 15 to 19 nucleotides but a 17 nucleotide spacer is favored. Promoter sequences, although not identical to one another display “consensus”, a pattern of sequence conservation in which some nucleotides are preferred over others at any given position. The sequences of promoters and terminators vary because the precise sequence of a promoter determines if it is a strong or a weak promoter, which in itself provides a mechanism for regulation of gene expression. Evolution and the mutations accumulated at each promoter site have contributed to “building” this highly-efficient mechanism. Transcriptional Elongation: Transcription occurs only in the 5’ to 3’ direction, just like DNA synthesis, thus only the DNA that is oriented in the 3’ to 5’ orientation can serve as a template for transcription. Thus genes can be encoded on both strands but reading in opposite directions:  EUKARYOTES have three separate RNA POLYMERASES: Eukaryotic cells require more than one RNA POL directed by multiple transcription factors, rather than a single σ factor to mediate transcription. Eukaryotic cells have three distinct RNA POLYMERASES each in charge of transcribing a particular set of genes. RNA POL I and RNA POL III transcribe genes whose terminal products are ribosomal subunits, tRNA, and small nuclear RNA. RNA POL II transcribes ALL PROTEIN CODING GENES. We will focus on RNA POL II in the following discussion.  RNA POL II of EUKARYOTES is similar to RNA POL of PROKARYOTES : Bacterial RNA POL is shown in green, behind is eukaryotic RNA POL II with additional regions shown in gray. Eukaryotic RNA POL II is larger (12 subunits instead of 5). RNA POL II is similar to bacterial RNA POL, except that it requires a whole set of accessory proteins called TRANSCRIPTION FACTORS (TFs) to initiate transcription. Transcription factors must assemble at the promoter for RNA POL to be able to begin transcription. Another difference between bacterial and eukaryotic RNA POL is that eukaryotic RNA POL must unwind the tightly packed chromatin structure in which DNA is condensed. Bacteria, not having a nucleus do not compact their DNA which is relatively loosely arranged in the cytoplasm of the cell. EUKARYOTIC TRANSCRIPTION: !" #$%&&'#()*+#,#-./0.*1.#2*#,#'34#-./0.*1.# 1,55.+#$4$4#(267#(8#9.,*#2:#,#$4$4;