LECTURE 3 Gene Transcription and RNA Modification (Chapter 12)

LECTURE 3 Gene Transcription and RNA Modification (Chapter 12)

INTRODUCTION The term gene has many definitions
For this class, a gene is a segment of DNA used to make a product that plays a functional role in the cell either an RNA or a polypeptide Transcription is the first step in gene expression

Court reporter transcribing court testimony
Words to Know Transcription: (Verb) The act or process of making a copy Example: Court reporter hears the witness speaking in English and types a written copy, in English, of the witness’ statements. Translation: Express the meaning of words or text in another language Dogma: A principle or set of principles laid down by an authority as incontrovertibly true Court reporter transcribing court testimony

TRANSCRIPTION In genetics, the term refers to the copying of a DNA sequence into an RNA sequence Only one strand is copied The structure of DNA is not altered as a result of this process It continues to store information and can be transcribed again and again and again

1. Check out 2. Make many copies of the same page 3. Return unaltered 4. Distribute and incite a riot!

Gene Expression Structural genes encode the amino acid sequence of a polypeptide Transcription of a structural gene produces messenger RNA, usually called mRNA The mRNA nucleotide sequence determines the amino acid sequence of a polypeptide during translation The synthesis of functional proteins determines an organisms traits This path from gene to trait is called the central dogma of genetics Refer to Figure 12.1

The central dogma of genetics
DNA replication: makes DNA copies that are transmitted from cell to cell and from parent to offspring. Gene Chromosomal DNA: stores information in units called genes. Transcription: produces an RNA copy of a gene. Messenger RNA: a temporary copy of a gene that contains information to make a polypeptide. Translation: produces a polypeptide using the information in mRNA. Polypeptide: becomes part of a functional protein that contributes to an organism's traits. Figure 12.1

Is this simplistic?

12.1 OVERVIEW OF TRANSCRIPTION
Gene expression is the overall process by which the information within a gene is used to produce a functional product which can, in concert with environmental factors, determine a trait Or: How does a book result in a riot?

The Stages of Transcription
Transcription occurs in three stages Initiation Elongation Termination These steps involve protein-DNA interactions Proteins such as RNA polymerase interact with DNA sequences

Transcription Figure 12.3 DNA of a gene Promoter Terminator
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA of a gene Transcription Promoter Terminator Initiation: The promoter functions as a recognition site for transcription factors (not shown). The transcription factor(s) enables RNA polymerase to bind to the promoter. Following binding, the DNA is denatured into a bubble known as the open complex. 5′ end of growing RNA transcript Open complex RNA polymerase Elongation/synthesis of the RNA transcript: RNA polymerase slides along the DNA in an open complex to synthesize RNA. Termination: A terminator is reached that causes RNA polymerase and the RNA transcript to dissociate from the DNA. Completed RNA transcript RNA polymerase Figure 12.3

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RNA Transcripts Have Different Functions
Once they are made, RNA transcripts play different functional roles Refer to Table 12.1 Well over 90% of all genes are structural genes which are transcribed into mRNA Final functional products are polypeptides The other RNA molecules in Table 12.1 are never translated Final functional products are RNA molecules

RNA Transcripts Have Different Functions
The RNA transcripts from nonstructural genes are not translated They do have various important cellular functions They can still confer traits In some cases, the RNA transcript becomes part of a complex that contains protein subunits For example Ribosomes Spliceosomes Signal recognition particles

You don’t need to memorize this slide – however, note how many different types of functional RNA molecules exist and how many different types of functions they perform!

12.2 TRANSCRIPTION IN BACTERIA
Our molecular understanding of gene transcription came from studies involving bacteria and bacteriophages Indeed, much of our knowledge comes from studies of a single bacterium E. coli, of course In this section we will examine the three steps of transcription as they occur in bacteria

Promoters Promoters are DNA sequences that “promote” gene expression
More precisely, they direct the exact location for the initiation of transcription 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 12.4

Figure 12.4 The conventional numbering system of promoters
Bases preceding the start site are numbered in a negative direction Most of the promoter region is labeled with negative numbers There is no base numbered 0 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Coding strand Transcriptional start site Promoter region –35 sequence 16 –18 bp –10 sequence +1 5′ 3′ T T C T T T G A A A A A A A A G A A A C T T T T T T 3′ 5′ Template strand 5′ 3′ Bases to the right are numbered in a positive direction RNA A Transcription Figure The conventional numbering system of promoters

Sequence elements that play a key role in transcription
The promoter may span a large region, but specific short sequence elements are particularly critical for promoter recognition and activity level Sequence elements that play a key role in transcription Coding strand Transcriptional start site Promoter region –35 sequence 16 –18 bp –10 sequence +1 5′ 3′ T T C T T T G A A A A A A A A G A A A C T T T T T T 3′ 5′ Template strand Sometimes termed the Pribnow box, after its discoverer 5′ 3′ RNA A Transcription Figure The conventional numbering system of promoters

Initiation of Bacterial Transcription
RNA polymerase is the enzyme that catalyzes the synthesis of RNA In E. coli, the RNA polymerase holoenzyme is composed of Core enzyme Five subunits = a2bb’ Sigma factor One subunit = s These subunits play distinct functional roles

Initiation of Bacterial Transcription
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 A region within the sigma factor that contains a helix-turn-helix structure is involved in a tighter binding to the DNA Refer to Figure 12.6

Binding of factor protein to DNA double helix
Amino acids within the a helices hydrogen bond with bases in the -35 and -10 promoter sequences Turn α helices binding to the major groove Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. HELIX Figure 12.6

A short RNA strand is made within the open complex
The binding of the RNA polymerase to the promoter forms the closed complex Then, the open complex is formed when the TATAAT box in the -10 region 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 This is known as the elongation phase

Figure 12.7 RNA polymerase Promotor region σ factor –35 –10
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. RNA polymerase Promotor region σ factor –35 –10 After sliding along the DNA, σ factor recognizes a promoter, and RNA polymerase holoenzyme forms a closed complex. RNA polymerase holoenzyme –35 –10 Closed complex An open complex is formed, and a short RNA is made. –35 –10 Open complex σ factor is released, and the core enzyme is able to proceed down the DNA. RNA polymerase core enzyme –35 –10 σ factor Figure 12.7 RNA transcript

In class skit Characters: Character Played By
A shy female college student A cute dude A helpful friend Dr. Ballard

Elongation in Bacterial Transcription
The RNA transcript is synthesized during the elongation stage The DNA strand used as a template for RNA synthesis is termed the template 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

In transcription, RNA polymerase reads only one strand of the DNA
It reads the template strand It moves along the template strand 3’ to 5’ The RNA polymerase simultaneously makes a RNA copy of the template strand’s complementary partner The partner strand is called the coding strand The new mRNA molecule is made in the 5’ to 3’ direction The orientation and sequence of the mRNA is identical to the coding strand (except U’s for T’s)

Which is the template strand? Which is the coding strand?

Elongation in Bacterial Transcription
The open complex formed by the action of RNA polymerase is about 17 bases long Behind the open complex, the DNA rewinds back into a double helix On average, the rate of RNA synthesis is about 43 nucleotides per second! Figure 12.8 depicts the key points in the synthesis of an RNA transcript

Similar to the synthesis of DNA via DNA polymerase
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.s Coding strand T A U C G C A T A G C G 5′ 3′ Coding strand Template strand 3′ Rewinding of DNA Template strand RNA polymerase Open complex Unwinding of DNA Direction of transcription RNA 5′ 3′ RNA–DNA hybrid region 5′ Nucleotide being added to the 3′ end of the RNA Nucleoside triphosphates Similar to the synthesis of DNA via DNA polymerase Key points: • RNA polymerase slides along the DNA, creating an open complex as it moves. • The DNA strand known as the template strand is used to make a complementary copy of RNA as an RNA–DNA hybrid. • RNA polymerase moves along the template strand in a 3′ to 5′ direction, and RNA is synthesized in a 5′ to 3′ direction using nucleoside triphosphates as precursors. Pyrophosphate is released (not shown). Figure 12.8 • The complementarity rule is the same as the AT/GC rule except that U is substituted for T in the RNA.

Termination of Bacterial Transcription
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 E. coli has two different mechanisms for termination 1. rho-dependent termination Requires a protein known as r (rho) 2. rho-independent termination Does not require r

Rho protein is a helicase
5′ 3′ Terminator rut RNA polymerase reaches the terminator. A stem-loop causes RNA polymerase to pause. Stem-loop RNA polymerase pauses due to its interaction with the stem-loop structure. ρ protein catches up to the open complex and separates the RNA-DNA hybrid. ρ recognition site (rut) ρ recognition site in RNA ρ protein binds to the rut site in RNA and moves toward the 3′ end. ρ protein Rho protein is a helicase rho utilization site r-dependent termination Figure 12.10

r-independent termination is facilitated by two sequences in the RNA
1. A uracil-rich sequence located at the 3’ end of the RNA 2. A stem-loop structure upstream of the uracil-rich sequence Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. U-rich RNA in the RNA-DNA hybrid 5′ Stem-loop that causes RNA polymerase to pause NusA Stabilizes the RNA pol pausing While RNA polymerase pauses, the U-rich sequence is not able to hold the RNA-DNA hybrid together. Termination occurs. URNA-ADNA hydrogen bonds are relatively weak Terminator No protein is required to physically remove the RNA from the DNA This type of termination is also called intrinsic 5′ U U U U 3′ r-independent termination Figure 12.11

12.3 TRANSCRIPTION IN EUKARYOTES
Many of the basic features of gene transcription are very similar in bacteria and eukaryotes However, gene transcription in eukaryotes is more complex Larger, more complex cells (organelles) Added cellular complexity means more genes that encode proteins are required Multicellularity adds another level of regulation express genes only in the correct cells at the proper time

Eukaryotic RNA Polymerases
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 gene And the 5S rRNA gene

Sequences of Eukaryotic Structural Genes
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 Regulatory elements TATA box Transcriptional start site Refer to Figure 12.13

regulatory elements such
Figure 12.13 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Core promoter Transcriptional start site Usually an adenine TATA box Coding-strand sequences: TATAAA Py2CAPy5 Common location for regulatory elements such as GC and CAAT boxes –100 –50 –25 +1 DNA Transcription The core promoter is relatively short It consists of the TATA box and transcriptional start site 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

regulatory elements such
Figure 12.13 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Core promoter Transcriptional start site TATA box Coding-strand sequences: TATAAA Py2CAPy5 Common location for regulatory elements such as GC and CAAT boxes –100 –50 –25 +1 DNA Transcription Regulatory elements are short DNA sequences that affect the binding of RNA polymerase to the promoter Transcription factors (proteins) bind to these elements and influence the rate of transcription There are two types of regulatory elements Enhancers Stimulate transcription Silencers Inhibit transcription They vary widely in their locations but are often found in the –50 to –100 region

Sequences of Eukaryotic Structural Genes
Factors that control gene expression can be divided into two types, based on their “location” cis-acting elements DNA sequences that exert their effect only over a particular gene Example: TATA box, enhancers and silencers trans-acting elements Regulatory proteins that bind to such DNA sequences

RNA Polymerase II and its Transcription Factors
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 (we won’t go over this) Figure shows the assembly of transcription factors and RNA polymerase II at the TATA box

Figure 12.14 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. TFIID binds to the TATA box. TFIID is a complex of proteins that includes the TATA-binding protein (TBP) and several TBP-associated factors (TAFs). TFIID You don’t need to memorize the binding order but you should know that several different general transcription factors must bind in order to recruit RNA polymerase to the promoter and start its action TATA box TFIIB binds to TFIID. TFIID TFIIB TFIIB acts as a bridge to bind RNA polymerase II and TFIIF. TFIID TFIIB TFIIF RNA polymerase II TFIIE and TFIIH bind to RNA polymerase II to form a preinitiation or closed complex. A closed complex TFIID Preinitiation complex TFIIB TFIIF TFIIH TFIIE TFIIH acts as a helicase to form an open complex. TFIIH also phosphorylates the CTD domain of RNA polymerase II. CTD phosphorylation breaks the contact between TFIIB and RNA polymerase II. TFIIB, TFIIE, and TFIIH are released. RNA pol II can now proceed to the elongation stage Released after the open complex is formed TFIID TFIIF Open complex TFIIB TFIIE TFIIH PO4 CTD domain of RNA polymerase II PO4 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

RNA Pol II transcriptional termination
Pre-mRNAs are modified by cleavage near their 3’ end with subsequent attachment of a string of adenines Transcription terminates 500 to 2000 nucleotides downstream from the poly A signal There are two models for termination Further research is needed to determine if either, or both are correct (we won’t cover this)

12.4 RNA MODIFICATION Analysis of bacterial genes in the 1960s and 1970 revealed the following: The sequence of DNA in the coding strand corresponds to the sequence of nucleotides in the mRNA The sequence of codons in the mRNA provides the instructions for the sequence of amino acids 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

12.4 RNA MODIFICATION 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

12.4 RNA MODIFICATION 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

Focus your attention here

Splicing Three different splicing mechanisms have been identified
Group I intron splicing Group II intron splicing Spliceosome (we’ll focus on this mechanism) All three cases involve Removal of the intron RNA Linkage of the exon RNA by a phosphodiester bond

In eukaryotes, the transcription of structural genes produces a long transcript known as pre-mRNA
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. Intron removed via spliceosome (very common in eukaryotes) Intron Spliceosome P O A O CH2 H 2′ 2′ H H Exon 1 O Exon 2 5′ O H P 3′ P A O O CH2 H 2 2 H H O O P P 3′ 5′ 3′ OH P O A O CH2 H 2′ 2′ H H O O P P 5′ 3′ mRNA (c) Pre-mRNA Figure 12.20

Pre-mRNA Splicing 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 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

The pre-mRNA splicing mechanism is shown in Figure 12.22
Intron RNA is defined by particular sequences within the intron and at the intron-exon boundaries The consensus sequences for the splicing of mammalian pre-mRNA are shown in Figure 12.21 Corresponds to the boxed adenine in Figure 12.22 Sequences shown in bold are highly conserved Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Exon Intron Exon 5′ UACUUAUCC A/CGGU Pu AGUA Py12N Py AGG 3′ 5′ splice site Branch site 3′ splice site Figure 12.21 Serve as recognition sites for the binding of the spliceosome The pre-mRNA splicing mechanism is shown in Figure 12.22

Intron loops out and exons brought closer together
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Exon 1 Exon 2 5′ GU A AG 3′ Branch site 5′ splice site 3′ splice site U1 binds to 5′ splice site. U2 binds to branch site. U1 snRNP U2 snRNP A 5′ 3′ U4/U6 and U5 trimer binds. Intron loops out and exons are brought closer together. Intron loops out and exons brought closer together A U2 U4/U6 snRNP U1 U5 snRNP 5′ 3′ Figure 12.22

Intron will be degraded and the snRNPs used again
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5′ splice site is cut. 5′ end of intron is connected to the A in the branch site to form a lariat. U1 and U4 are released. Cleavage may be catalyzed by snRNA molecules within U2 and U6 U4 U1 U2 A U6 U5 5′ 3′ 3′ splice site is cut. Exon 1 is connected to exon 2. The intron (in the form of a lariat) is released along with U2, U5, and U6. The intron will be degraded. Intron will be degraded and the snRNPs used again U2 A Intron plus U2, U5, and U6 U6 U5 5′ Exon 1 Two connected exons Exon 2 3′ Figure 12.22

Intron Advantage? One benefit of genes with introns is a phenomenon called alternative splicing A pre-mRNA with multiple introns can be spliced in different ways This will generate mature mRNAs with different combinations of exons This variation in splicing can occur in different cell types or during different stages of development

Intron Advantage? 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

Alternative Splicing One very important biological advantage of introns in eukaryotes is the phenomenon of alternative splicing Alternative splicing refers to the phenomenon that pre-mRNA can be spliced in more than one way Alternatively splicing produces two or more polypeptides with different amino acid sequences In most cases, large sections of the coding regions are the same, resulting in alternative versions of a protein that have similar functions Nevertheless, there will be enough differences in amino acid sequences to provide each polypeptide with its own unique characteristics

Alternative Splicing The degree of splicing and alternative splicing varies greatly among different species Baker’s yeast contains about 6,300 genes ~ 300 (i.e., 5%) encode mRNAs that are spliced Only a few of these 300 have been shown to be alternatively spliced Humans contain ~ 25,000 genes Most of these encode mRNAs that are spliced It is estimated that about 70% are alternatively spliced Note: Certain mRNAs can be alternatively spliced to produce dozens of different mRNAs

Alternative Splicing Figure considers an example of alternative splicing for a gene that encodes a-tropomyosin This protein functions in the regulation of cell contraction It is found in Smooth muscle cells (uterus and small intestine) Striated muscle cells (cardiac and skeletal muscle) Also in many types of nonmuscle cells at low levels The different cells of a multicellular organism regulate contractibility in subtly different ways One way to accomplish this is to produce different forms of a-tropomyosin by alternative splicing

Found in the mature mRNA from all cell types
Not found in all mature mRNAs Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Intron α-tropomyosin pre-mRNA Exon 5′ 1 2 3 4 5 6 7 8 9 1 1 1 1 2 1 3 1 4 3′ Constitutive exons Alternative splicing Alternative exons 5′ 1 2 4 5 6 8 9 1 1 4 3′ Smooth muscle cells or 5′ 1 3 4 5 6 8 9 1 1 1 1 2 3′ Striated muscle cells These alternatively spliced versions of a-tropomyosin vary in function to meet the needs of the cell type in which they are found Figure Alternative ways that the rat a-tropomyosin pre-mRNA can be spliced

Capping Most mature mRNAs have a 7-methylguanosine covalently attached at their 5’ end This event is known as capping Capping occurs as the pre-mRNA is being synthesized by RNA pol II Usually when the transcript is only 20 to 25 bases long As shown in Figure 12.23, capping is a three-step process

Figure 12.23 5′ O O O Base O– P O P O P O CH2 O O– O– O– H H H H O OH
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5′ O O O Base O O O Base O– P O P O P O CH2 O– O– O– O H H O– P O P O P O CH2 H H O OH O O P O- O– O– O– O CH2 H H H H O OH O P O– O CH2 Rest of mRNA 3′ RNA 5′-triphosphatase removes a phosphate. Pi O O Base O– P O P O CH2 O O– O– H H H H O O H O P O– O CH2 Rest of mRNA Guanylyltransferase hydrolyzes GTP. The GMP is attached to the 5′ end, and PPi is released. Figure 12.23 PPi

Figure 12.23 H O N NH2 N N N H OH O O O Base H HO CH2 O P O P O P O
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. H O N NH2 N N N H OH O O O Base H HO CH2 O P O P O P O CH2 O H O– O– O– O H H H H H O OH O P O– O H CH2 O N Rest of mRNA NH2 CH3 Methyltransferase attaches a methyl group. N + N N H OH O O O Base H HO CH2 O P O P O P O CH2 O H O– O– O– O H H H H H 7-methylguanosine cap O OH O P O– O CH2 Rest of mRNA Figure 12.23

Capping The 7-methylguanosine cap structure is recognized by cap-binding proteins Cap-binding proteins play roles in the Movement of some RNAs into the cytoplasm Early stages of translation Splicing of introns

Tailing 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 12.24

Polyadenylation signal sequence
Figure 12.24 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Polyadenylation signal sequence 5′ 3′ AAUAAA Endonuclease cleavage occurs about 20 nucleotides downstream from the AAUAAA sequence. Consensus sequence in higher eukaryotes 5′ AAUAAA PolyA-polymerase adds adenine nucleotides to the 3′ end. 5′ AAAAAAAAAAAA.... 3′ AAUAAA Appears to be important in the stability of mRNA and the translation of the polypeptide PolyA tail Length varies between species From a few dozen adenines to several hundred

LECTURE 3 Gene Transcription and RNA Modification (Chapter 12)

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LECTURE 3 Gene Transcription and RNA Modification (Chapter 12)

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