Principles of BiochemistryFourth Edition

Chapter 21Transcription and RNA Processing

Copyright © 2006 Pearson Prentice Hall, Inc.

Horton • Moran • Scrimgeour • Perry • Rawn

Chapter 21 - Transcription and RNA Processing

• Gene - a DNA sequence that is transcribed (includes genes that do not encode proteins)

• “Housekeeping genes” encode proteins or RNA essential for normal activities of the cell (e.g. enzymes in basic metabolic pathways, tRNAs and rRNAs)

• Biological information flow from DNA to proteins is irreversible

Fig 21.1 Biological information flow

21.1 Types of RNA

(1) Transfer RNA (tRNA)•Carries amino acids to translation

machinery•Very stable molecules

(2) Ribosomal RNA (rRNA) •Makes up much of the ribosome•Very stable, majority of cellular RNA

(3) Messenger RNA (mRNA) •Encodes message from DNA to ribosomes•Rapidly degraded by nucleases

Table 21.1

21.2 RNA Polymerase

• RNA polymerase (RNA pol) catalyzes DNA-directed RNA synthesis (transcription)

• RNA pol is core of a larger transcription complex

• Complex assembles at one end of a gene when transcription is initiated

• DNA is continuously unwound as RNA pol catalyzes a processive elongation of RNA chain

A. RNA Polymerase Is an Oligomeric Protein

• Core enzyme: ’participates in many of the transcription reactions

• Subunit contains part of polymerase site

• ’ Subunit contributes to DNA binding

• Subunits are scaffolding and interact with other proteins that regulate

transcription

• Role not well characterized

• Subunit role in initiation of transcription

Table 21.2

Fig 21.2 Structure of T. aquaticus RNA pol core enzyme

B. The Chain Elongation Reaction

• Mechanism almost identical to that for DNA polymerase

• Growing RNA chain is base-paired to DNA template strand

• Incoming ribonucleotide triphosphates (RTPs) form correct H bonds to template

• New phosphodiester bond formed, PPi released

• Catalyzes polymerization in 5’ 3’ direction

• Is highly processive, and thermodynamically assisted by PPi hydrolysis

• Incoming RTPs: UTP, GTP, ATP, CTP

• Growing single-stranded RNA released

• Adds 30-85 nucleotides/sec (~ 1/10th rate of DNA replication)

RNA polymerase reaction

Fig 21.3

• RNA polymerase reaction

Fig 21.3

• RNA polymerase reaction

Fig 21.3 (cont)

21.3 Transcription Initiation

• Transcription complex assembles at an initiation site (DNA promoter region)

• Short stretch of RNA is synthesized

• Operon: a transcription unit in which several genes are often cotranscribed in prokaryotes

• Eukaryotic genes each have their own promoter

Fig 21.4 Transcription of E. coli ribosomal RNA genes

A. Genes Have a 5’ → 3’ Orientation

• Convention for double-stranded DNA:Coding strand (top) is written: 5’ → 3’Template strand (bottom) is written: 3’ → 5’

• Gene is transcribed from 5’ end to the 3’ end

• Template strand of DNA is copied from the 3’ end to the 5’ end

• Growth of RNA chain proceeds 5’ → 3’

Fig 21.5 Orientation of a gene

B. Transcription Complex Assembles at a Promoter

• Consensus sequences are found upstream from transcription start sites

• DNA-binding proteins bind to promoter sequences (prokaryotes and eukaryotes) and direct RNA pol to the promoter site

• The subunit of prokaryotic RNA polymerases is required for promoter recognition and formation of the complex

Fig 21.6 Promoter sequences from 10 bacteriophage and bacterial genes

E. coli Promoter

(1) TATA box (-10 bp upstream from transcription start site (rich in A/T bp)

(2) -35 region (-35 bp upstream) from start site

• A subunit also required (e.g. 70)

• Strong promoters match consensus sequence closely (operons transcribed efficiently)

• Weak promoters match consensus sequences poorly (operons transcribed infrequently)

C. The Subunit Recognizes the Promoter

• Subunit increases the affinity of the core polymerase for specific promoter sequences

• Subunit also decreases the affinity of the core polymerase for nonpromoter regions

• Core polymerase (no subunit) binds DNA nonspecifically

Table 21.3

D. RNA Polymerase Changes Conformation

• Unwinding of DNA at the initiation site requires a conformational change

• RNA pol (R) and promoter (P) shift from:

(RPc) closed complex (DNA double stranded)

(RPo) open complex (18 bp DNA unwound)(forms transcription bubble)

Fig 21.7 Initiation of transcription in E. coli

Fig 21.7 Initiation of transcription in E. coli(two slides)

Fig 21.7 (continued)

AnimationsAnimations

• Transcription

21.4 Transcription Termination

• Only certain regions of DNA are transcribed

• Transcription complexes assemble at promoters and disassemble at the 3’ end of genes at specific termination sequences

• Two types of termination sequences: (1) Unstable elongation complex (2) Rho-dependent termination

Pause Sites

• Pause sites - regions of the gene where the rate of elongation slows down (10 to 100-fold) or stops temporarily

• Transcription termination often occurs here

• G-C- rich regions are more difficult to separate than A-T rich regions and may be pause sites

• Pause is exaggerated when newly transcribed RNA can form a hairpin

Fig 21.8 Formation of an RNA hairpin

Rho-dependent Termination Sites

• Rho triggers disassembly of the transcription complex at some pause sites

• Rho binds to ssRNA chain, destabilizing the RNA-DNA hybrid and terminating transcription

Fig 21.9

• Rho-dependent termination of transcription (E. coli)

• RNA pol is stalled at pause site

• Rho binds to new RNA, destabilizes RNA-DNA hybrid

21.5 Transcription in Eukaryotes

A. Eukaryotic RNA Polymerases

• Three different RNA polymerases transcribe nuclear genes

• Other RNA polymerases found in mitochondria and chloroplasts

• Table 21.4 (next slide) summarizes these RNA polymerases

Fig 21.10 RNA polymerase II from S. cerevisiae (yeast)

Fig 21.11 A Generic Eukaryotic Promoter

B. Eukaryotic Transcription Factors

• Same reactions as prokaryotic transcription

• More complicated assembly of machinery

• Binding of RNA polymerase to promoters requires a number of initiation transcription factors (TFs)

Table 21.5

RNA polymerase II

• Transcribes all protein-encoding genes and some small RNA encoding genes

• Protein-encoding RNA synthesized by RNA pol II is called mRNA precursor (or hnRNA)

• 20%-40% of all cellular RNA synthesis

• General transcription factors interact directly with RNA pol II and control initiation at class II genes

Fig 21.12 A. thaliana TATA-binding protein (TBP) (blue) bound to DNA

Fig 21.13 RNA polymerase II holoenzyme complex bound to a promoter

B. The Role of Chromatin in Eukaryotic Transcription

• Status of chromatin affects gene

expression

• Chromatin status is modulated by

– Post-transcriptional modification of

histones, e.g. methlylation or acetylation

21.6 Transcription of Genes Is Regulated

• Expression of housekeeping genes is constitutive (constitutive genes)

• These genes usually have strong promoters and are efficiently and continuously transcribed

• Housekeeping genes whose products are required at low levels have weak promoters and are infrequently transcribed

• Regulated genes are expressed at different levels under different conditions

Role of regulatory proteins in transcription initiation

• Regulatory proteins bind to specific DNA sequences and control initiation of transcription

• Repressors - regulatory proteins that prevent transcription of a negatively regulated gene

• Negatively regulated genes can only be transcribed in the absence of the repressor

• Activators - regulatory proteins that activate transcription of a positively regulated gene

Inducers and Corepressors

• Repressors and activators are often allosteric proteins modified by ligand binding

• Inducers - ligands that bind to and inactivate repressors

• Corepressors - ligands that bind to and activate repressors

• Four general strategies for regulating transcription (Figure 21.14, next four slides)

Fig. 21.14

• Strategies for regulating transcription initiation by regulatory proteins

AnimationsAnimations

• Transcriptional Regulation

21.7 The lac Operon, an Example of Negative and Positive Regulation

• E. coli can use lactose and other -galactosides as a carbon source

• Uptake and catabolism of -galactosides requires three proteins

• When glucose is present these proteins are synthesized in limited amounts

• When glucose levels are low these proteins are synthesized in larger amounts

The Proteins of E. coli -Galactoside Metabolism

(1) Lactose permease (lacY gene product) - transporter for uptake of -galactosides

(2) -Galactosidase (lacZ gene product) - hydrolyzes -galactosides to hexoses

(3) Thiogalactoside transacetylase (lacA gene product) - acetylates (add CH3CO-)

nonmetabolizable -galactosides prior to elimination from the cell

The lac Operon

• Three genes - lacZ, lacY and lacA form an operon that is transcribed from a single promoter

• Resulting large mRNA molecule contains three separate protein-coding regions (3 genes)

• Operons with multiple protein-coding regions are common in prokaryotes but not eukaryotes

A. lac Repressor Blocks Transcription

• lac Operon expression is controlled by the lac repressor protein

• lac Repressor is encoded by the lacI gene, located upstream from the lac operon

• lac Repressor binds simultaneously to 2 repressor-binding sites (operators)

• Operator (O1) is adjacent to the promoter, (O2) is within the coding region of lacZ

Fig 21.15 Organization of the genes for proteins required to metabolize lactose

• Three coding regions of lac operon are cotranscribed from the Plac promoter

• lacI is under the control of the P promoter

Repressor-operator Interactions

• The lac repressor binds simultaneously to both operators O1 and O2

• This causes the DNA to form a stable loop which blocks initiation of transcription

• -Galactosides act as inducers to cause the repressor to dissociate from the operon allowing transcription to continue

Fig 21.16 Electron micrographs of DNA loops

Fig 21.17 Binding of lac repressor to the lac operon

• Tetrameric lac repressor interacts simultaneously with two sites near the lac promoter

• DNA loop forms

• RNA pol can still bind to the promoter

Allolactose Inducer for the lac Operon

• In the absence of lactose, the lac repressor blocks expression of the lac operon

• Several -galactosides can act as inducers to start gene transcription

• In the presence of lactose, allolactose is the inducer

• -Galactosidase converts lactose to allolactose

Fig 21.18

• Formation of allolactose from lactose

B. The Structure of the lac Repressor

Left: End-on view

Right: Side view, lac repressor -helix in the major groove

C. cAMP Regulatory Protein Activates Transcription

• The lac operon is transcribed maximally when -galactosides are the only carbon source

• When glucose is present transcription is reduced 50-fold, termed catabolite repression

• A weak promoter in these operons is promoted to a stronger one by an activator (in the absence of glucose)

Activation of the lac Operon

• cAMP regulatory (or receptor) protein (CRP) is the activator

• CRP is also known as catabolite activator protein (CAP)

• In the presence of cAMP, CRP binds near the promoters of more than 30 genes

• RNA polymerase is activated and the rate of transcription activation is increased

Fig 21.20

• Activation of transcription initiation at the lac promoter by CRP-cAMP

Fig 21.20 (cont)

Fig 21.21 cAMP production

• In the absence of glucose, enzyme III (EIII) transfers a phosphate group to adenylate cyclase leading to CRP-cAMP increases

• CRP-cAMP activates transcription of other genes

Fig 21.22

• Conformational changes in CRP caused by cAMP binding

• -Helices of each monomer of the cAMP-CRP dimer fit into major groove of DNA

Fig 21.23 Structure of CRP-cAMP and DNA complex

• Both subunits have a cAMP bound at the allosteric site

• Each subunit has an -helix in DNA major groove

21.8 Posttranscriptional Modification of RNA

• RNA processing

• Removal of nucleotides from primary RNA transcripts

• Addition of nucleotides with sequences not encoded by corresponding genes

• Covalent modification of certain bases

21.8 Posttranscriptional Modification of RNA

• Mature tRNA molecules are generated in both prokaryotes and eukaryotes by processing the primary transcripts, termed RNA processing.

• In prokaryotes, the primary transcripts often contain several tRNA precursors

• Ribonucleases (RNases) cleave the large primary transcripts to their mature lengths

Fig 21.24

• Summary of prokaryotic tRNA processing (three slides)

Fig 21.25 Examples of covalent modification of nucleotides in tRNA molecules

Fig 21.25 Examples of covalent modification of nucleotides in tRNA molecules

Fig 21.25 (cont)

Fig 21.25 (cont)

B. Ribosomal RNA Processing

• Ribosomal RNA in all organisms are produced as large primary transcripts that require processing

• Processing includes methylation and cleavage by endonucleases

• Prokaryotic rRNA primary transcripts ~30S

• Contain one copy each: 16S, 23S, 5S rRNA

Fig 21.26 Endonucleolytic cleavage of rRNA precursors in E. coli

21.9 Eukaryotic mRNA Processing

• In prokaryotes the primary mRNA transcript is translated directly

• In eukaryotes transcription occurs in the nucleus, translation in the cytoplasm

• Eukaryotic mRNA is processed in the nucleus without interfering with translation

• In some mRNA, pieces are removed from the middle and the ends joined (splicing)

A. Eukaryotic mRNA Molecules Have Modified Ends

• All eukaryotic mRNA precursors undergo modifications to increase their stability and make them better substrates for translation

• Ends are modified so they are no longer susceptible to exonuclease degradation

• The 5’ ends are modified (formation of a cap) before the mRNA precursors are completely synthesized

Fig 21.27

Formation of a cap at the 5’ end of a eukaryotic mRNA precursor

phosphohydrolase

Fig 21.27

phosphohydrolase

Fig 21.27 (cont)

Fig 21.27 (cont)

Guanylate base is methylated at N-7

methylase

Fig 21.27 (cont)

2- Hydroxyl groups of last two riboses may also be methylated

methylase

Poly A tails at the 3’ ends of mRNA precursors

• Eukaryotic mRNA precursors are also modified at their 3’ ends (formation of a poly A tail)

• A poly A polymerase adds up to 250 adenylate residues to the 3’ end of the mRNA precursor

• This poly A tail is progressively shortened by 3’ exonucleases

• The poly A tail increases the time required for nucleases to reach the coding region

Fig 21.28 Polyadenylation of a eukaryotic mRNA precursor

Fig 21.28 (cont)

CPSF: a cleavage and polyadenylation specificity factor

Fig 21.28 (cont)

B. Some Eukaryotic mRNA Precursors are Spliced

• Introns - internal sequences that are removed from the primary RNA transcript

• Exons - sequences that are present in the primary transcript and the mature mRNA

• Splice sites - junctions of the introns and exons where mRNA precursor is cut and joined

Fig 21.29 Triose phosphate isomerase gene (nine exons and eight introns)

Fig 21.29 (cont)

• Protein 3-dimensional structure showing parts of protein encoded by each exon

Fig 21.30 Consensus sequences at splice sites in vertebrates

Y: pyrimidine (U or C)R: purine (A or G)N: any nucleotide

The Spliceosome

• The spliceosome is a large RNA-protein complex which catalyzes splicing reactions

• It contains 45 proteins and 5 small nuclear RNA (snRNA) molecules (“snurps”)

Fig 21.31 Intron removal in mRNA precursors

Fig 21.31 (cont)

Fig 21.31 (cont)

Fig 21.32 Formation of a spliceosome

** Each snRNP contains one or two snRNA plus a number of proteins.

Fig 21.32 (cont)

Fig 21.32 (cont)