Biological Science Chapter 17: Transcription, RNA Processing, and Translation - PDF

Biological Science Seventh Edition Chapter 17 Transcription RNA Processing, and T...

Biological Science Seventh Edition Chapter 17 Transcription RNA Processing, and Translation Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved You should now be able to: 1. Define control elements and explain how they influence transcription 2. Explain the process of transcription in bacteria and eukaryotes 3. Explain the role of promoters, enzymes and transcription factors in transcription 4. Describe the steps in mRNA processing. 5. List the steps involved in translation and all of the factors involved. 6. Describe the structure of the ribosome. Small and large subunit, A, P and E site. 7. Understand protein folding and the players involved in the process. Introduction to Transcription, R N A Processing, and Translation Proteins serve various roles in cells A cell builds the proteins it needs from instructions encoded in its genome: – Transcribe DNA into RNA – Translate mRNA into protein Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved 17.1 An Overview of Transcription Transcription Is the Synthesis of RN A from a D N A Template RNA polymerases synthesize an RNA version of the instructions stored in DNA: – Uses ribonucleoside triphosphates (NTPs) – Matches complementary bases to one strand of DNA Only one strand of DNA is the template strand: – Other strand is the non-template, or coding strand: ▪ Matches the sequence of the mRNA (except U for T) Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved An Overview of Transcription Like DNA polymerases: – RNA polymerases perform template-directed synthesis in the 5′ → 3′ direction Unlike DNA polymerases: – RNA polymerases do not require a primer to begin transcription Bacteria have one RNA polymerase Eukaryotes have at least three distinct types: – RNA polymerase I, II and III Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Initiation: How Does Transcription Begin in Bacteria? Initiation is the first phase of transcription RNA polymerase cannot initiate transcription on its own: – In bacteria, sigma protein must bind to it first – RNA polymerase and sigma form a holoenzyme: ▪ Sigma recognizes sites, called promoters, where transcription begins ▪ RNA polymerase transcribes genes and is The Bacterial R N A Polymerase Holoenzyme the core enzyme Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Bacterial Promoters Bacterial promoters are 40–50 base pairs long: – The −10 box is a TATAAT sequence ~10 bases upstream of the transcription start site: ▪ Downstream means in the same direction that RNA polymerase moves ▪ Upstream is the opposite direction – The −35 box is a TTGACA sequence about 35 bases upstream of the transcription start site Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Events Inside the Holoenzyme (1 of 2) Transcription begins when the sigma part of the holoenzyme complex binds to the −35 and −10 boxes Sigma can bind in only one orientation: – Thus, promoter orientation determines which D N A strand will be used as the template – Also determines direction RNA polymerase will move Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Events Inside the Holoenzyme (2 of 2) RNA polymerase opens the DNA double helix: – Creates a transcription bubble – The template strand is threaded through the RNA polymerase active site Incoming NTPs enter a channel in the enzyme and diffuse to the active site Complementary NTPs pair with complementary DNA bases and polymerization begins Sigma Orients the R N A Polymerase on DN A during the Initiation of Transcription Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Elongation and Termination in Bacteria During elongation, RNA polymerase reads the DNA template: – Nucleotides are added to the 3’ end of the RNA Termination occurs when RNA polymerase transcribes a transcription-termination signal: Transcription in Bacteria – Codes for RNA that Terminates When an R N A Hairpin Forms forms a hairpin structure – Causes the RNA Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Transcription in Eukaryotes Eukaryotic transcription has several differences: – Three RNA polymerases – Larger, more diverse promoters, including TATA box – General transcription factors recognize promoters, rather than sigma proteins – At termination, a poly(A) signal is transcribed rather than a hairpin, and the R NA downstream is cut – Transcription occurs in the nucleus, and translation occurs in the cytoplasm Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Web Activity: RNA Synthesis Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved 17.2 RNA Processing in Eukaryotes In bacteria, transcription produces fully functional R NAs In eukaryotes, the initial product of transcription is an immature primary transcript or pre-mRNA Primary transcripts must undergo RNA processing before they can be translated Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved The Startling Discovery of Split Eukaryotic Genes The Discovery of Introns In 1977, Roberts and Sharp showed that protein-coding genes contained noncoding DNA: – Viral DNA was heated to form single-stranded DNA, then incubated with mRNA produced by the virus – Loops formed along the hybrid molecules – Stretches of DNA are not present in the mature mRNA Introns are not present in the final mRNA Exons are present in the final m RNA Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved RNA Splicing (1 of 2) Primary RNA transcripts contain exons and introns Introns are removed by splicing: – Catalyzed by small nuclear ribonucleoproteins (snRNPs): ▪ Made of small nuclear RNAs (snRNAs) and protein ▪ Form a complex called a spliceosome Splicing allows different m RNAs and proteins to be produced from a single gene- alternative splicing Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved RNA Splicing (2 of 2) Four steps to splicing: 1. snRNPs bind to the 5′ exon–intron and 3’ intron- exon boundaries and to an A near the end of the intron 2. Other snRNPs join the complex to form a spliceosome 3. The intron forms a single-stranded stem plus a loop (lariat) with A as its connecting point 4. The lariat is cut out and the two exons are linked. The intron is degraded Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Figure 17.6 During Splicing, Introns Are Cut Out of the Primary Transcript Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Adding Caps and Tails to Transcripts In Eukaryotes, a Cap and a Tail Are Added to mRNAs Pre-mRNA s are processed by two additional events: – 5 cap: modified guanine nucleotide that enables ribosomes to bind and protect from degradation – Poly(A) tail: 100–250 adenine nucleotides needed for translation and protects from degradation; added after cleaving 3’ end After splicing and addition of the cap and tail, the product is a mature mRNA Mature mRNA s contain untranslated regions (UTRs) at both ends Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved 17.3 An Introduction to Translation The sequence of mRNA bases is converted to an amino acid sequence A complex set of steps are involved Translation is carried out with the use of ribosomes, mRNA, and tRNAs Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Ribosomes Are the Site of Protein Synthesis Britten and colleagues performed a pulse-chase experiment to show ribosomes are the site of protein synthesis A pulse of radioactive sulfur is incorporated into amino acids, followed by a chase of unlabeled sulfur This showed that proteins are synthesized at ribosomes, then released Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved  An Overview of Translation Transcription and Translation Can Be Coupled, or Occur Simultaneously, in Bacteria In bacteria, ribosomes begin translating an mRNA before transcription is complete: – Multiple ribosomes attached to an mRNA form a polyribosome – Many copies of a protein are produced from one mRNA In eukaryotes, transcription and translation are separated: – mRNAs are synthesized and processed in the nucleus – Mature mRNA s are transported to the cytoplasm for Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved How Does mRNA Specify Amino Acids? Two Hypotheses for How Codons Interact with Amino Acids There were two hypotheses regarding how m RNA determined an amino acid sequence: 1. mRNA codons and amino acids interact directly. 2. Crick proposed an adapter molecule holding the amino acid in place while interacting with a codon Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved 17.4 the Structure and Function of Transfer RNA The adapter molecule used in translation is called transfer RNA (tRNA): – An aminoacyl tRNA is a tRNA linked to its amino acid – Amino acids are transferred from tRNAs to a growing polypeptide Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved What is the Structure of tRNA s? tRNA s are relatively short: 75–95 nucleotides long They can form secondary structures by folding into a stem-and-loop A CCA sequence at the 3′ end is the amino acid binding site The loop at the opposite end contains the anticodon: – Has a sequence of three nucleotides – Can base-pair with the The Structure of an Aminoacyl Transfer RNA mRNA codon Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved How Are Amino Acids Attached to tRNAs? ATP is required to attach tRNA to an amino acid Aminoacyl-tRNA synthetases “charge” the tRNA: – Catalyze the addition of amino acids to tRNAs There are 20 amino acids: – Each has a different aminoacyl tRNA synthetase Aminoacyl-t RN A Synthetases Couple the Appropriate – For each amino acid, there is one or Amino Acid to the more tRNAs Appropriate t RN A Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved How Many tRNAs Are There? There are 61 different codons but only about 40 t R NAs in most cells Crick proposed wobble pairing as a solution: – The anticodon’s third position can form a nonstandard base pair One tRNA is able to read more than one codon Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved 17.5 Ribosome Structure and Function in Translation Ribosomes contain many proteins and ribosomal RNA (rRNA) Ribosomes can be separated into two subunits: 1. The small subunit holds the mRNA in place 2. The large subunit is where peptide bonds form During translation, three tRNAs line up within the ribosome Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Ribosome Structure and Function in Translation (1 of 3) Ribosomes Contain Three tRN A Binding Sites The tRNAs fit into three sites in the ribosome, bound to corresponding mRNA codons: 1. A site (acceptor or aminoacyl)—tRNA carries an amino acid 2. P site (peptidyl)—holds growing peptide chain 3. E site (exit)—tRNA s without amino acids exit the Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Ribosome Structure and Function in Translation (2 of 3) The ribosome synthesizes proteins in a three-step sequence: 1. An aminoacyl tRNA enters the A site; remains if there is a codon-anticodon match 2. A peptide bond forms between the amino acid on the A-site tRNA and the polypeptide on the P-site tRN A 3. The ribosome moves down the mRNA by one codon and all three tRNAs move down one ▪ The tRNA in the E site exits position: ▪ The A site is available for another t RNA to bind Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Ribosome Structure and Function in Translation (3 of 3) The protein grows by one amino acid with each repeat of the three steps Amino acids are always added to the carboxyl end (C-terminus) of the polypeptide Translation has three phases: 1. Initiation 2. Elongation 3. Termination Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Initiating Translation (1 of 2) The initiation phase of translation begins near the AUG start codon In bacteria, the small ribosomal subunit binds to the ribosome binding site, or Shine–Dalgarno sequence, of the mRNA: – About 6 bases upstream from the start codon – Mediated by initiation factors The first tRNA is called the initiator tRNA: – It carries a modified methionine (f-Met) in bacteria Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Initiating Translation (2 of 2) Translation initiation is a three-step process in bacteria: 1. The mRNA binds to a small ribosomal subunit 2. The initiator tRNA bearing f-Met binds to the start codon 3. The large ribosomal subunit binds so that the initiator tRNA is in the P site Translation is now ready to begin Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Figure 17.14 Initiation Requires Binding of Initiator tRNA to mRNA, and Assembly of the Ribosome Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Elongation: Extending the Polypeptide At the start of elongation: – The initiator tRNA is in the P site – The E and A sites are empty An aminoacyl tRNA binds to the codon in the A site Amino acids at the P and A sites are in the ribosome’s active site, where peptide bond formation occurs The amino acid on the P-site tRNA is transferred to the amino acid on the A-site tRNA Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Is the Ribosome an Enzyme or a Ribozyme? The active site of the ribosome is entirely ribosomal RNA Ribosomal RNA catalyzes peptide bond formation Therefore, the ribosome is a ribozyme This supports the RNA world hypothesis Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Moving Down the mRNA (1 of 2) Translocation occurs when the ribosome slides one codon toward the 3′ end of the mRNA: – Elongation factors help move the ribosome Translocation accomplishes three things: 1. The uncharged tRNA from the P site moves into the E site and is ejected from the ribosome 2. The tRNA attached to the growing protein moves into the P site 3. Opens the A site to expose a new codon, which is available to accept a new aminoacyl t RNA Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Figure 17.15 Elongation Extends the Polypeptide Chain—Part 1 Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Moving Down the mRNA (2 of 2) The three steps of translocation repeat at each codon along the mRNA: 1. Arrival of the aminoacyl tRNA 2. Peptide bond formation 3. Translocation Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Figure 17.15 Elongation Extends the Polypeptide Chain—Part 2 Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Terminating Translation Termination occurs when the A site encounters a stop codon A protein called a release factor enters the A site: – Resembles tRNAs in size and shape – Hydrolyzes the bond linking the P-site tRNA to the polypeptide chain The newly synthesized polypeptide, tRNAs, and ribosomal subunits separate from the m RNA Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Figure 17.16 Termination Occurs When a Release Factor Binds to a Stop Codon Encountered by the Ribosome Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Table 17.1 Transcription, R NA Processing, and Translation in Bacteria and Eukaryotes Process Bacteria Eukaryotes Transcription RNA polymerase(s) One Three; each produces a different class of R NA Promoter structure Typically contains a -35 box and a More variable and larger; often includes a TATA −10 box box about -30 from the transcription start site Proteins that associate with promoter Sigma; different versions of sigma Many general transcription factors bind to different promoters RNA processing Rare Extensive; several processing steps occur in the nucleus before R NA is exported to the cytoplasm: (1) enzyme-catalyzed addition of 5′ cap on mRNAs, (2) splicing (intron removal) by the spliceosome to produce m RNA, and (3) enzyme- catalyzed addition of 3′ poly(A) tail on m RNAs Translation (initiation, Initiation and termination less Initiation and termination more complex; elongation elongation, and complex; elongation similar to similar to bacteria termination) eukaryotes Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Web Activity: Synthesizing Proteins Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Polypeptides Are Modified after Translation Most proteins go through an extensive series of processing steps before they are functional: – Called post-translational modification These steps take place in various locations in a cell Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Polypeptide Folding and Chemical Modifications Folding determines a protein’s shape The shape of a protein determines its function: – Molecular chaperones guide and speed up protein folding Sugars or lipid groups may be added to proteins Enzymes may add a phosphate group to a protein to alter its activity Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved Figure 17.17 The Major Steps of Gene Expression in a Eukaryotic Cell Copyright © 2020, 2017, 2014 Pearson Education, Inc. All Rights Reserved