12_18_lecture(1).pptx

Chapter 18 Gene Expression: I. The Genetic Code and Transcription Lectures by Kathleen Fitzpatrick © 2016 Pearson Education, Inc. Simon Fraser University Gene Expression: I. The Genetic Code and Transcription  The coded information of DNA is used to guide production of RNA and protein molecules...

Chapter 18 Gene Expression: I. The Genetic Code and Transcription Lectures by Kathleen Fitzpatrick © 2016 Pearson Education, Inc. Simon Fraser University Gene Expression: I. The Genetic Code and Transcription  The coded information of DNA is used to guide production of RNA and protein molecules  Transcription refers to RNA synthesis using DNA as a template  Translation is the synthesis of protein using the information in the RNA © 2016 Pearson Education, Inc. Transcription and Translation Involve Many of the Same Components in Bacteria and Eukaryotes  Messenger RNA, mRNA, is RNA that is translated into protein  Ribosomal RNA, rRNA, is an integral component of the ribosome  Transfer RNA, tRNA, molecules serve as intermediaries, bringing amino acids to the ribosome  The latter two function in translation © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. Where Transcription and Translation Occur Differs in Bacteria and Eukaryotes  Because bacteria do not have a nuclear envelope, translation of mRNA can begin before its transcription is completed  The compartmentalization of eukaryotic cells leads to spatial separation of transcription and translation © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. In Some Cases RNA Is Reverse Transcribed into DNA  In certain cases, RNA can serve as a template for DNA synthesis  This process of reverse transcription is catalyzed by the enzyme reverse transcriptase  Viruses that carry out reverse transcription are called retroviruses © 2016 Pearson Education, Inc. Retroviruses  Two copies of the RNA genome. Each RNA molecule has an reverse transcriptase  Once inside the cell, reverse transcriptase catalyzes synthesis of a DNA  The resulting double-stranded DNA then enters the nucleus and integrates into the genome of the host © 2016 Pearson Education, Inc. Retrotransposons  Reverse transcription also occurs in normal eukaryotic cells in the absence of viral infection  Much of it involves DNA elements called retrotransposons  These use reverse transcription to move from one site to another within a genome © 2016 Pearson Education, Inc. Retrotransposon Movement  Transposition begins with transcription of the retrotransposon DNA followed by translation of the resulting RNA  This produces a protein with reverse transcriptase and endonuclease activities  The retrotransposon RNA and protein then bind chromosomal DNA in a new location © 2016 Pearson Education, Inc. Retrotransposon Movement (continued)  The endonuclease cuts one of the DNA strands  The reverse transcriptase uses the retrotransposon RNA as a template to make a DNA copy that is then integrated into the target site © 2016 Pearson Education, Inc. Alu Sequences  Retrotransposons can attain high copy numbers within a genome despite transposing rarely  Alu sequences are 300 bp long and do not encode a reverse transcriptase  But using reverse transposase from elsewhere in the genome, they have increased their copy number in humans and other primates  In the human genome, about 1 million Alu sequences represent about 11% of the total DNA © 2016 Pearson Education, Inc. The Genetic Code  The relationship between the DNA base sequence and the linear order of amino acids in the protein products is based on a set of rules known as the genetic code © 2016 Pearson Education, Inc. Gene Function Is Complicated  Most eukaryotic genes contain noncoding sequences among the coding regions of the gene  Coding sequences can be read in various combinations, each coding for a unique polypeptide chain; this is called alternative splicing  Some types of genes encode functional RNAs  Genes are thus defined as functional units of DNA that encode one or more polypeptides or functional RNA © 2016 Pearson Education, Inc. The Genetic Code Is a Triplet Code  There are four DNA bases and 20 amino acids  A triplet code, in which combinations of three bases specify amino acids, would have 64 possible combinations, more than enough for all 20 amino acids © 2016 Pearson Education, Inc. Frameshift Mutations  The gene is written in a language of three-letter words  Inserting or deleting a nucleotide (indel mutations) causes the rest of the sequence to be read out of phase—this is a shift in the reading frame  Mutations that cause insertion or deletion of a nucleotide are thus called frameshift mutations © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. The Genetic Code Is Degenerate and Nonoverlapping  There are 64 combinations of nucleotide triplets and only 20 amino acids  The genetic code is a degenerate code, particular amino acid can be specified by more than one triplet  It is also nonoverlapping; the reading frame advances three nucleotides at a time © 2016 Pearson Education, Inc. Messenger RNA Guides the Synthesis of Polypeptide Chains  The genetic code refers to the order of nucleotides in the mRNA molecules that direct protein synthesis  mRNA is transcribed from DNA similarly to how DNA is replicated, but with two differences © 2016 Pearson Education, Inc. Differences Between mRNA Synthesis and DNA Replication  In mRNA synthesis, only one DNA strand is copied, called the template strand; the other strand is called the coding strand because it is similar to the mRNA sequence  In mRNA synthesis, a uracil base (U) is used instead of thymine © 2016 Pearson Education, Inc. Of the 64 Possible Codons in Messenger RNA, 61 Code for Amino Acids  All 64 codons are used in the translation of mRNA  61 of them specify a specific amino acids  One of them, AUG, plays a role as a start codon  The remaining three (UAA, UAG, UGA) are stop codons © 2016 Pearson Education, Inc. The Genetic Code Is Unambiguous and Degenerate  Every codon has one meaning only—the genetic code is unambiguous  It is also degenerate—many of the amino acids are specified by more than one codon  With a degenerate code, most mutations cause codon changes and a changed amino acid © 2016 Pearson Education, Inc. The Genetic Code Is (Nearly) Universal  Except for a few cases, all organisms use the same basic genetic code  In the case of mitochondria and a few bacteria, the genetic code differs in several ways  For example, AGA is a stop codon in mammalian mitochondria, and in some organisms codons specify nonstandard amino acids © 2016 Pearson Education, Inc. 18.2 Mechanisms of Transcription  RNA is chemically similar to DNA but contains ribose instead of deoxyribose  It also has the base uracil instead of thymine and is usually single stranded  The fundamental principles of transcription were first elucidated in bacteria © 2016 Pearson Education, Inc. Transcription Involves Four Stages: Binding, Initiation, Elongation, and Termination  The DNA that gives rise to one RNA molecule is called the transcription unit  Transcription begins when RNA polymerase binds to a promoter sequence (1), triggering local unwinding of the double helix  RNA polymerase then initiates synthesis of RNA using one DNA strand as a template (2) © 2016 Pearson Education, Inc. Steps of RNA Synthesis (continued)  After initiation, the RNA polymerase moves along the DNA template, unwinding the helix and elongating the RNA (3)  The unwound DNA is called a transcription bubble  Eventually, RNA polymerase dissociates from the DNA template, leading to termination of synthesis and release of the RNA molecule © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. Bacterial Transcription Involves σ Factor Binding, Initiation, Elongation, and Termination © 2016 Pearson Education, Inc. Binding of RNA Polymerase to a Promoter Sequence  RNA polymerase binds to a DNA promoter site  The terms upstream and downstream refer to sequences located toward the 5′ or 3′ end of the transcription unit, respectively  The promoter is upstream of the transcribed sequence © 2016 Pearson Education, Inc. Techniques for Identifying Promoter Sequences  Promoter sequences were initially identified by DNA footprinting and electrophoretic mobility shift assays  More recently, chromatin immunoprecipitation (ChIP) has been used to assess binding of proteins to certain genomic DNA sequences in eukaryotes  Even more recently!! © 2016 Pearson Education, Inc. Essential Sequences in a Typical Bacterial Promoter  The transcription start site is almost always a purine and usually an adenine  About 10 bp upstream of the start site is the sequence TATAAT, called the –10 sequence  At or near the –35 position is the sequence TTGACA, called the –35 sequence © 2016 Pearson Education, Inc. Consensus Sequences and UP Elements  The consensus sequence of a promoter (and other conserved sequences) are those nucleotides most commonly found at a particular position in the sequence  Many bacterial promoters also contain upstream (UP) elements  These are associated with particular strong promoters © 2016 Pearson Education, Inc. RNA Polymerase Subunits  Bacterial cells have a single kind of RNA polymerase that synthesizes all major classes of RNA  RNA polymerase is a large protein consisting of two α subunits, two β subunits, and a dissociable subunit called the sigma (σ) factor © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. RNA Polymerase  The core enzyme lacks the sigma subunit and can carry out RNA synthesis  However the holoenzyme (including all the subunits) is required to ensure initiation at all sites within the DNA molecule  The sigma subunit promotes binding of RNA polymerase at the –35 and –10 elements © 2016 Pearson Education, Inc. Initiation of RNA Synthesis  Initiation of RNA synthesis takes place once the DNA is unwound  One of the DNA strands serves as a template for RNA synthesis, using incoming ribonucleoside triphosphate molecules (NTPs) that are complementary to the template strand  RNA polymerase catalyzes the formation of a phosphodiester bond between the NTPs © 2016 Pearson Education, Inc. RNA Polymerases and RNA Initiation  RNA polymerase initiates synthesis until the chain is as much as nine nucleotides in length  This abortive synthesis continues producing and releasing short pieces of RNA while the polymerase remains attached to the promoter  It pulls some downstream DNA into its interior, a process called scrunching the DNA © 2016 Pearson Education, Inc. The End of the Initiation Phase  Eventually a piece of RNA ten nucleotides or longer is produced  This weakens the interaction of the RNA polymerase with the promoter, allowing it to escape the promoter  The RNA polymerase releases the sigma factor and the polymerase moves along the DNA to transcribe an elongating RNA © 2016 Pearson Education, Inc. Elongation of the RNA Chain  Chain elongation continues as RNA polymerase moves along the DNA molecule  The RNA is elongated in the 5′ to 3′ direction, with each new nucleotide added to the 3′ end  As the polymerase moves along the DNA strand, the double helix ahead of the polymerase is unwound, and the DNA behind it is rewound into a double helix © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. RNA Proofreading  RNA backtracking  When an incorrect nucleotide is incorporated, the polymerase backs up slightly and the incorrect nucleotide and the previous one are removed  This is RNA proofreading; occasional errors in RNA molecules are not as critical as errors in DNA replication © 2016 Pearson Education, Inc. Termination of RNA Synthesis  Elongation of the RNA chain proceeds until the RNA polymerase copies a sequence called the termination signal  There are two types of termination signals based on whether or not they require a protein called the rho (ρ) factor © 2016 Pearson Education, Inc. Types of Termination Signal  RNA molecules that terminate without the rho factor contain a short GC-rich sequence followed by several U’s  The GC region in the RNA forms a hairpin loop pulling the RNA molecule away from the DNA  Then the weaker bonds between the U’s and the A’s of the template strand break, releasing the RNA © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. Types of Termination Signal (continued)  RNA molecules that don’t form the GC-rich hairpin require the rho factor for termination  The rho factor is an ATP-dependent unwinding enzyme moving along the RNA molecule toward the 3′ end and unwinding it from the DNA template as it proceeds  Eventually it will interrupt the RNA polymerase © 2016 Pearson Education, Inc. Transcription in Eukaryotic Cells Has Additional Complexity Compared with Prokaryotes  Eukaryotic transcription involves the same four stages as prokaryotic, but there are several important differences  Three different RNA polymerases transcribe one or more different classes of RNA  Eukaryotic promoters are more varied than bacterial ones; some are even located downstream of the gene © 2016 Pearson Education, Inc. Eukaryotic Transcription  Eukaryotic transcription differs from that of prokaryotes  RNA polymerases in eukaryotes require additional proteins called transcription factors, some of which must bind before the RNA polymerase can bind  Protein-protein interactions play a prominent role in eukaryotic transcription © 2016 Pearson Education, Inc. Eukaryotic Transcription (continued)  Eukaryotic transcription differs from that of prokaryotes  RNA cleavage is more important than termination of transcription in determining the 3′ end of the transcript  Newly forming RNA molecules undergo RNA processing, chemical modification during and after transcription © 2016 Pearson Education, Inc. RNA Polymerase I, II, and III Carry Out Transcription in the Eukaryotic Nucleus  There are three RNA polymerases in the nucleus, designated RNA polymerases I, II, and III  These differ in their location in the nucleus and the types of RNA they synthesize © 2016 Pearson Education, Inc. The RNA Polymerases  RNA polymerase I, in the nucleolus, synthesizes an RNA molecule that is a precursor for four types of rRNA  RNA polymerase II is found in nucleoplasm and synthesizes mRNA; the molecules are found in clusters called transcription factories, where active genes congregate to be transcribed  RNA polymerase III, in the nucleoplasm, synthesizes a variety of small RNAs, including tRNA, and the 5S rRNA © 2016 Pearson Education, Inc. Three Classes of Promoters Are Found in Eukaryotic Nuclear Genes, One for Each Type of RNA Polymerase  Eukaryotic promoters are varied but can be grouped into three categories  The promoter used by RNA polymerase I has two parts  The core promoter is the smallest set of DNA sequences that initiates transcription © 2016 Pearson Education, Inc. The Upstream Control Element  The core promoter is sufficient for initiation of transcription  However, transcription occurs more efficiently in the presence of an upstream control element, a fairly long sequence similar to the core promoter © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. The Promoter for RNA Polymerase II  At least four types of DNA sequences are involved in core promoter function 1. A short initiator sequence surrounds the transcription start point 2. The TATA box, a consensus sequence of TATA followed by two to three A’s, is located about 25 nucleotides upstream of the start point © 2016 Pearson Education, Inc. The Promoter for RNA Polymerase II (continued)  At least four types of DNA sequences are involved in core promoter function (continued) 3. The TFIIB recognition element (BRE) is located slightly upstream of the TATA box 4. The downstream promoter element (DPE) is located about 30 nucleotides downstream from the start point © 2016 Pearson Education, Inc. Two Types of Core Promoters  The four elements are organized into two general types of core promoters  TATA-driven promoters contain an initiator (Inr) sequence and a TATA box with or without an associated BRE  DPE-driven promoters contain DPE and Inr sequences but no TATA box or BRE © 2016 Pearson Education, Inc. Additional Control Elements  Core promoters are capable of driving only a basal (low) level of transcription  Additional short sequences upstream (upstream control elements) improve the promoter’s efficiency  Some are common to many different genes, such as the CAAT box and the GC box © 2016 Pearson Education, Inc. Upstream Control Elements  The location of upstream control elements varies from gene to gene  Those within 100–200 nucleotides of the start point are called proximal control elements  Those farther away are called enhancer elements © 2016 Pearson Education, Inc. Promoters for RNA Polymerase III  RNA polymerase III uses promoters that are entirely downstream of the start point  In both 5S RNA and tRNA, the promoters are different, but both consensus sequences fall into two blocks of about 10 bp each  tRNA has box A and box B; rRNA has box A and box C © 2016 Pearson Education, Inc. General Transcription Factors Are Involved in the Transcription of All Nuclear Genes  A general transcription factor is always required for RNA polymerase binding to promoters  Eukaryotes have many TFs that bind the promoter in a defined order starting with TFIID  Eventually, a large complex of proteins forms a preinitiation complex on the promoter © 2016 Pearson Education, Inc. General Transcription Factors  TFIIH possesses helicase activity that unwinds DNA and protein kinase activity that phosphorylates the RNA polymerase II C-terminal domain (CTD)  This releases RNA polymerase II from the transcription factors so it can begin RNA synthesis  TFIID recognizes and binds DNA because of its TATA-binding protein (TBP) subunit © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc. Other Proteins Needed for Transcription  Besides general transcription factors and RNA polymerase II, several other kinds of proteins are needed  Some open chromatin structure to make DNA accessible to RNA polymerase  Others are regulatory factors that bind upstream control elements and recruit coactivator proteins © 2016 Pearson Education, Inc. Elongation, Termination, and RNA Cleavage Are Involved in Completing Eukaryotic RNA Synthesis  After initiation, RNA polymerases move along the DNA and synthesize a complementary RNA  Termination is governed by signals that differ for each type of RNA polymerase  Transcription by RNA polymerase I is terminated by a protein that recognizes an 18-nucleotide signal in the growing RNA chain © 2016 Pearson Education, Inc. Termination of Transcription  For RNA polymerase III, termination signals include a short run of U’s, and no protein factors are required for their recognition  For RNA polymerase II, transcripts are cleaved at a specific site before transcription ceases  The cleavage site is 10–35 nucleotides downstream of a AAUAAA sequence in the RNA © 2016 Pearson Education, Inc. Polyadenylation  The cleavage site of polymerase II transcripts is also the site for addition of a poly(A) tail  This is a string of adenine nucleotides added to the 3′ end of most eukaryotic mRNAs © 2016 Pearson Education, Inc.