PowerPoint Presentation Materials to accompany Genetics: Analysis and Principles Robert J. Brooker Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display CHAPTER 12 GENE TRANSCRIPTION AND RNA MODIFICATION

Transcription literally means the act or process of making a copy DNA sequence to RNA sequence 1. DNA sequences provide the underlying information Signals for the start and end of transcription 2. Proteins recognize these sequences and carry out the process Other proteins modify the RNA transcript to make it functionally active 12-2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

At the molecular level, a gene is a transcriptional unit During gene expression, different types of base sequences perform different roles Figure 12.1 shows a common organization of sequences within a bacterial gene and its transcript Gene Expression Requires Base Sequences 12-4

Figure Bacterial mRNA may be polycistronic, which means it encodes two or more polypeptides Start codon: specifies the first amino acid in a protein sequence, usually a formylmethionine (in bacteria) or a methionine (in eukaryotes) Signals the end of protein synthesis Bacterial transcriptional unit

A eukaryotic gene and its transcript

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The strand that is actually transcribed is termed the template strand The opposite strand is called the coding strand or the sense strand The base sequence is identical to the RNA transcript Except for the substitution of uracil in RNA for thymine in DNA Gene Expression Requires Base Sequences 12-6

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Transcription occurs in three stages Initiation Elongation Termination These steps involve protein-DNA interactions Proteins such as RNA polymerase interact with DNA sequences The Stages of Transcription 12-7

12-8 The promoter functions as a recognition site for transcription factors The transcription factors enable RNA polymerase to bind to the promoter forming a closed promoter complex Following binding, the DNA is denatured into a bubble known as the open promoter complex, or simply an open complex Initiation Elongation RNA polymerase slides along the DNA in an open complex to synthesize the RNA transcript Termination A termination signal is reached that causes RNA polymerase to dissociated from the DNA Figure 12.2

The initiation of transcription at a eukaryotic promoter

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display A structural gene is a one that encodes a polypeptide When such genes are transcribed, the product is an RNA transcript called messenger RNA (mRNA) Other RNA transcripts becomes part of a complex that contains protein subunits For example Ribosomes Spliceosomes Signal recognition particles RNA Transcripts Have Different Functions 12-10

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Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12.2 TRANSCRIPTION IN BACTERIA 12-12

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Promoters are DNA sequences that “promote” gene expression Rate Start site Promoters are typically located just upstream of the site where transcription of a gene actually begins The bases in a promoter sequence are numbered in relation to the transcription start site Refer to Figure 12.3 Promoters 12-13

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 12.3 The conventional numbering system of promoters Bases preceding this are numbered in a negative direction There is no base numbered 0 Bases to the right are numbered in a positive direction Sometimes termed the Pribnow box, after its discoverer Sequence elements that play a key role in transcription

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 12.4 Examples of –35 and –10 sequences within a variety of bacterial promoters The most commonly occurring bases For many bacterial genes, there is a good correlation between the rate of RNA transcription and the degree of agreement with the consensus sequences

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display RNA polymerase is the enzyme that catalyzes the synthesis of RNA In E. coli, the RNA polymerase holoenzyme is composed of Core enzyme Four subunits =  2  ’ Sigma factor One subunit =  These subunits play distinct functional roles Initiation of Bacterial Transcription 12-16

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The RNA polymerase holoenzyme binds loosely to the DNA It then scans along the DNA, until it encounters a promoter region When it does, the sigma factor recognizes both the –35 and –10 regions Initiation of Bacterial Transcription 12-17

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 12.5 Amino acids within the  helices hydrogen bond with bases in the promoter sequence elements

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The binding of the RNA polymerase to the promoter forms the closed complex Then, the open complex is formed when the TATAAT box is unwound A short RNA strand is made within the open complex The sigma factor is released at this point This marks the end of initiation The core enzyme now slides down the DNA to synthesize an RNA strand 12-19

12-20 Figure 12.6 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The RNA transcript is synthesized during the elongation step The DNA strand used as a template for RNA synthesis is termed the template or noncoding strand The opposite DNA strand is called the coding strand It has the same base sequence as the RNA transcript Except that T in DNA corresponds to U in RNA Elongation in Bacterial Transcription 12-21

12-23 Similar to the synthesis of DNA via DNA polymerase Figure 12.7

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Termination is the end of RNA synthesis It occurs when the short RNA-DNA hybrid of the open complex is forced to separate This releases the newly made RNA as well as the RNA polymerase Termination of Bacterial Transcription 12-24

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Nuclear DNA is transcribed by three different RNA polymerases RNA pol I Transcribes all rRNA genes (except for the 5S rRNA) RNA pol II Transcribes all structural genes Thus, synthesizes all mRNAs Transcribes some snRNA genes RNA pol III Transcribes all tRNA genes And the 5S rRNA gene Eukaryotic RNA Polymerases 12-29

Fig a(TE Art) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Structure of a bacterial RNA polymerase Structure of a eukaryotic RNA polymerase II

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Eukaryotic promoter sequences are more variable and often more complex than those of bacteria For structural genes, at least three features are found in most promoters Transcriptional start site TATA box Regulatory elements Refer to Figure Sequences of Eukaryotic Structural Genes 12-31

12-32 Usually an adenine The core promoter is relatively short It consists of the TATA box Important in determining the precise start point for transcription The core promoter by itself produces a low level of transcription This is termed basal transcription Figure Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

12-33 Figure Regulatory elements affect the binding of RNA polymerase to the promoter They are of two types Enhancers Stimulate transcription Silencers Inhibit transcription They vary in their locations but are often found in the –50 to –100 region Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Usually an adenine

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display cis-acting elements DNA sequences that exert their effect only on nearby genes Example: TATA box, enhancers and silencers trans-acting elements Regulatory proteins that bind to such DNA sequences Transcription factors Sequences of Eukaryotic Structural Genes 12-34

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Three categories of proteins are required for basal transcription to occur at the promoter RNA polymerase II Five different proteins called general transcription factors (GTFs) A protein complex called mediator Figure shows the assembly of transcription factors and RNA polymerase II at the TATA box RNA Polymerase II and its Transcription Factors 12-35

12-36 Figure Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

12-37 Figure Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display A closed complex TFIIH plays a major role in the formation of the open complex It has several subunits that perform different functions One subunit hydrolyzes ATP and phosphorylates a domain in RNA pol II known as the carboxyl terminal domain (CTD) This releases the contact between TFIIB and RNA pol II Other subunits act as helicases Promote the formation of the open complex Released after the open complex is formed RNA pol II can now proceed to the elongation stage

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Basal transcription apparatus RNA pol II + the five GTFs The third component for transcription is a large protein complex termed mediator It mediates interactions between RNA pol II and various regulatory transcription factors Its subunit composition is complex and variable 12-38

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Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The compaction of DNA to form chromatin can be an obstacle to the transcription pocess Most transcription occurs in interphase Then, chromatin is found in 30 nm fibers that are organized into radial loop domains Within the 30 nm fibers, the DNA is wound around histone octamers to form nucleosomes Chromatin Structure and Transcription 12-40

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The histone octamer is roughly five times smaller than the complex of RNA pol II and the GTFs The tight wrapping of DNA within the nucleosome inhibits the function of RNA pol To circumvent this problem, the chromatin structure is significantly loosened during transcription Two common mechanisms alter chromatin structure Chromatin Structure and Transcription 12-41

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 1. Covalent modification of histones Amino terminals of histones are modified in various ways Acetylation; phosphorylation; methylation Figure Adds acetyl groups, thereby loosening the interaction between histones and DNA Removes acetyl groups, thereby restoring a tighter interaction

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 2. ATP-dependent chromatin remodeling The energy of ATP is used to alter the structure of nucleosomes and thus make the DNA more accessible Figure Proteins are members of the SWI/SNF family Acronyms refer to the effects on yeast when these enzyme are defective Mutants in SWI are defective in mating type switching Mutants in SNF are sucrose non-fermenters These effects may significantly alter gene expression

Analysis of bacterial genes in the 1960s and 1970s revealed the following: The sequence of DNA in the coding strand corresponds to the sequence of nucleotides in the mRNA This in turn corresponds to the sequence of amino acid in the polypeptide This is termed the colinearity of gene expression Analysis of eukaryotic structural genes in the late 1970s revealed that they are not always colinear with their functional mRNAs Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12.4 RNA MODIFICATION 12-44

Instead, coding sequences, called exons, are interrupted by intervening sequences or introns Transcription produces the entire gene product Introns are later removed or excised Exons are connected together or spliced This phenomenon is termed RNA splicing It is a common genetic phenomenon in eukaryotes Occurs occasionally in bacteria as well Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12.4 RNA MODIFICATION 12-45

Aside from splicing, RNA transcripts can be modified in several ways For example Trimming of rRNA and tRNA transcripts 5’ Capping and 3’ polyA tailing of mRNA transcripts Refer to Table 12.3 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12.4 RNA MODIFICATION 12-46

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A eukaryotic gene and its transcript

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Many nonstructural genes are initially transcribed as a large RNA This large RNA transcript is enzymatically cleaved into smaller functional pieces Figure shows the processing of mammalian ribosomal RNA Trimming 12-48

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure Functional RNAs that are key in ribosome structure This processing occurs in the nucleolus

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure RNase P (Endonuclease) Endonuclease (RNase D) Found to contain both RNA and protein subunits However, RNA contains the catalytic ability Therefore, it is a ribozyme Covalently modified bases

In the late 1970s, several research groups investigated the presence of introns in eukaryotic structural genes One of these groups was led by Phillip Leder Leder used electron microscopy to identify introns in the  -globin gene It had been cloned earlier Leder used a strategy that involved hybridization Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Experiment 12A: Identification of Introns Via Microscopy 12-52

Double-stranded DNA of the cloned  -globin gene is first denatured Then mixed with mature  -globin mRNA The mRNA is complementary to the template strand of the DNA So the two will bind or hybridize to each other If the DNA is allowed to renature, this complex will prevent the reformation of the DNA double helix Refer to Figure Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Experiment 12A: Identification of Introns Via Microscopy 12-53

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure RNA displacement loop mRNA cannot hybridize to this region Because the intron has been spliced out from the mRNA

The Hypothesis The  -globin gene from the mouse contains one or more introns Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Testing the Hypothesis Refer to Figure

Figure

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The Data

Interpreting the Data Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Hybridization caused the formation of two R loops, separated by a double- stranded DNA region This suggests that the  -globin gene contains introns

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Three different splicing mechanisms have been identified Group I intron splicing Group II intron splicing Spliceosome All three cases involve Removal of the intron RNA Linkage of the exon RNA by a phosphodiester bond Splicing 12-59

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Splicing among group I and II introns is termed self-splicing Splicing does not require the aid of enzymes Instead the RNA itself functions as its own ribozyme Group I and II self-splicing can occur in vitro without the additional proteins 12-60

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 12.18

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure In eukaryotes, the transcription of structural genes, produces a long transcript known as pre-mRNA Also as heterogeneous nuclear RNA (hnRNA) This RNA is altered by splicing and other modifications, before it leaves the nucleus Splicing in this case requires the aid of a multicomponent structure known as the spliceosome

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Table 12.4 describes the occurrence of introns in genes of different species 12-63

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Most mature mRNAs have a 7-methyl guanosine covalently attached at their 5’ end This event is known as capping Cap-binding proteins play roles in the Movement of some RNAs into the cytoplasm Early stages of translation Splicing of introns Capping 12-64

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 12.19

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Most mature mRNAs have a string of adenine nucleotides at their 3’ ends This is termed the polyA tail The polyA tail is not encoded in the gene sequence It is added enzymatically after the gene is completely transcribed The attachment of the polyA tail is shown in Figure Tailing 12-68

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure Consensus sequence in higher eukaryotes Appears to be important in the stability of mRNA and the translation of the polypeptide Length varies between species From a few dozen adenines to several hundred

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The spliceosome is a large complex that splices pre-mRNA It is composed of several subunits known as snRNPs (pronounced “snurps”) Each snRNP contains small nuclear RNA and a set of proteins Pre-mRNA Splicing 12-70

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The subunits of a spliceosome carry out several functions 1. Bind to an intron sequence and precisely recognize the intron-exon boundaries 2. Hold the pre-mRNA in the correct configuration 3. Catalyze the chemical reactions that remove introns and covalently link exons Pre-mRNA Splicing 12-71

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure Intron RNA is defined by particular sequences within the intron and at the intro-exon boundaries The consensus sequences for the splicing of mammalian pre-mRNA are shown in Figure Sequences shown in bold are highly conserved Corresponds to the boxed adenine in Figure Serve as recognition sites for the binding of the spliceosome The pre-mRNA splicing mechanism is shown in Figure 12.22

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Intron loops out and exons brought closer together Figure 12.22

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure Intron will be degraded and the snRNPs used again

Alternative RNA splicing

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The biological advantage of alternative splicing is that two (or more) polypeptides can be derived from a single gene This allows an organism to carry fewer genes in its genome Intron Advantage? 12-76