Beruflich Dokumente
Kultur Dokumente
CHAPTER 5
Gene Expression: Transcription
Chapter 5 slide 1
1. Transcription, or gene expression, is regulated by gene regulatory elements associated with each gene.
2. DNA unwinds in the region next to the gene, due to RNA polymerase in prokaryotes and other proteins in eukaryotes. In both, RNA polymerase catalyzes transcription (Figure 5.1).
601 20000 Chapter 5 slide 3
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 4
3. RNA is transcribed 5-to-3. The template DNA strand is read 3-to-5. Its complementary DNA, the nontemplate strand, has the same polarity as the RNA.
Fig. 5.2 Chemical reaction involved in the RNA polymerase-catalyzed synthesis of RNA on a DNA template strand
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 6
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 8
4. Promoters in E. coli generally involve two DNA sequences, centered at -35 bp and -10 bp upstream from the +1 start site of transcription.
5. The common E. coli promoter that is used for most transcription has these consensus sequences:
a. For the -35 region the consensus is 5-TTGACA-3. b. For the -10 region (previously known as a Pribnow box), the consensus is 5-TATAAT-3.
6. Transcription initiation requires the RNA polymerase holoenzyme to bind to the promoter DNA sequence. Holoenzyme consists of:
a. Core enzyme of RNA polymerase, containing four polypeptides (two , one and one ). b. Sigma factor ().
7. Sigma factor binds the core enzyme, and confers ability to recognize promoters and initiate RNA synthesis. Without sigma, the core enzyme randomly binds DNA but does not transcribe it efficiently. 8. RNA polymerase holoenzyme binds promoter in two steps (Figure 5.4):
a. First, it loosely binds to the -35 sequence. b. Second, it binds tightly to the -10 sequence, untwisting about 17 bp of 601 20000 Chapter 5 slide 9 DNA at the site, and in position to begin transcription.
Fig. 5.4 Action of E. coli RNA polymerase in the initiation and elongation stages of transcription
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 10
9. Promoters often deviate from consensus. The associated genes will show different levels of transcription, corresponding with sigmas ability to recognize their sequences.
10. E. coli has several sigma factors with important roles in gene regulation. Each sigma can bind a molecule of core RNA polymerase and guide its choice of genes to transcribe. 11. Most E. coli genes have a 70 promoter, and 70 is usually the most abundant sigma factor in the cell. Other sigma factors may be produced in response to changing conditions. Examples of E. coli sigma factors:
a. 70 recognizes the sequence TTGACA at -35, and TATAAT at -10. b. 32 recognizes the sequence CCCCC at -39 and TATAAATA at -15. Sigma32 arises in response to heat shock and other forms of stress. c. 54 recognizes the sequence GTGGC at -26 and TTGCA at -14. Sigma54 arises in the response to heat shock and other forms of stress. d. 23 recognizes the sequence TATAATA at position -15. Sigma23 is present in cells infected with phage T4.
12. E. coli has additional sigma factors. Other bacterial species also have multiple sigma factors.
601 20000 Chapter 5 slide 11
2. Core enzyme untwists DNA helix locally, allowing a small region to denature. Newly synthesized RNA forms an RNA-DNA hybrid, but most of the transcript is displaced as the DNA helix reforms. The chain grows at 3050 nt/second.
3. RNA polymerase has two types of proofreading:
a. Similar to DNA polymerase editing, newly inserted nucleotide is removed by reversing synthesis reaction. b. b. Enzyme moves back one or more nucleotides, cleaves RNA, then resumes synthesis in
forward direction.
4. Terminator sequences are used to end transcription. In prokaryotes there are two types:
a. Rho-independent (-independent) or type I terminators have two-fold symmetry that would allow a hairpin loop to form (Figure 5.5). The palindrome is followed by 4-8U residues in the trasncript, and together these sequences cause termination, possibly because rapid hairpin formation destabilizes the RNA-DNA hybrid. b. Rho-dependent (-dependent) or type II terminators lack the poly(U) region, and many also lack the palindrome. The protein is required for termination. It has two domains, one binding RNA and the other binding ATP. ATP hydrolysis provides energy for to move along the transcript and destablize the RNA-DNA hybrid at the termination region.
601 20000
Chapter 5 slide 12
Fig. 5.5 Sequence of a -independent terminator and structure of the terminated RNA
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 13
Transcription in Eukaryotes
1. Prokaryotes contain only one RNA polymerase, which transcribes all RNA for the cell. 2. Eukaryotes have three different polymerases, each transcribing a different class of RNA. Processing of transcripts is also more complex in eukaryotes.
601 20000
Chapter 5 slide 14
2. Eukaryotic RNA polymerases are harder to study than the prokaryotic counterpart, because they are present at low concentrations. 3. All known eukaryotic RNA polymerases have multiple subunits. An example is yeast RNA pol II with 12 subunits, 5 of which are also in its RNA pol III (Figure 601 20000 Chapter 5 slide 15 5.6).
4. Various combinations of core and proximal elements are found near different genes. Promoter proximal elements are key to gene expression.
a. Activators, proteins important in transcription regulation, are recognized by promoter proximal elements. b. Housekeeping (used in all cell types for basic cellular functions) genes have common promoter proximal elements and are recognized by activator proteins found in all cells. Examples: i. Actin ii. Glucose-6-phosphate dehydrogenase c. Genes expressed only in some cell types or at particular times have promoter proximal elements recognized by activator proteins found only in specific cell types or times.
5. Enhancers are another cis-acting element. They are required for maximal transcription of a gene.
a. Enhancers are usually upstream of the transcription initiation site, but may also be downstream. They may modulate from a distance of thousands of base pairs away from the initiation site. b. Enhancers contain short sequence elements, some similar to promoter sequences.
c. Activators bind these sequences and other protein complexes form, bringing the enhancer complex close to the promoter and increasing transcription.
601 20000
Chapter 5 slide 17
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 18
6. Transcription initiation requires assembly of RNA polymerase II and binding of general transcription factors (GTFs) on the core promoter.
a. GTFs are needed for initiation by all three RNA polymerases. b. GTFs are numbered to match their corresponding RNA polymerase, and lettered in the order of discovery (e.g., TFIID was the fourth GTF discovered that works with RNA polymerase II). 7. Sequence of binding is not completely understood. a. Binding of GTFs and RNA pol II occurs in a set order in in vitro experiments (Figure 5.7) to produce the complete transcription initiation complex or preinitiation complex (PIC): b. The situation is less clear in vivo. Some data indicate that initiation complex forms before binding promoter. c. Transcription for eukaryotes is also complicated by the nucleosome organization of chromosomes.
601 20000
Chapter 5 slide 19
601 20000
Chapter 5 slide 20
Box Fig. 5.1 Sucrose density gradient centrifugation technique for separating and isolating RNA molecules in a mixture
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 21
Eukaryotic mRNAs
Animation: mRNA Production in Eukaryotes
b. The coding sequence, which specifies the amino acid sequence of the protein that will be produced during translation. It varies in length according to the size of the protein that it encodes.
c. The trailer sequence, or 3 untranslated region (3 UTR) also varies in length and contains information influencing the stability of the mRNA.
601 20000 Chapter 5 slide 22
Fig. 5.8 General structure of mRNA found in both prokaryotic and eukaryotic cells
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 23
Fig. 5.9 Processes for synthesis of functional mRNA in prokaryotes and eukaryotes
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 25
601 20000
Chapter 5 slide 26
5 and 3 Modifications
1. The newly made 5 end of the mRNA is modified by 5 capping. A capping enzyme adds a guanine, usually 7-methyl guanosine (m7G), to the 5 end using a 5-to-5 linkage. Sugars of the 2 adjacent nt are also methylated. The cap is used for ribosome binding to the mRNA during translation initiation. (Figure 5.10)
601 20000
Chapter 5 slide 27
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 28
d. PABII (poly(A) binding protein II) binds the poly(A) tail as it is produced.
601 20000
Chapter 5 slide 29
Fig. 5.11 Diagram of the 3 end formation of mRNA and the addition of the poly(A) tail
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 30
Introns
1. Removal of introns is necessary for mRNA maturation, as hnRNA (Heteronuclear RNA) becomes functional mRNA. 2. in Philip leders lab (1978) it was shown that the mouse -globin pre-mRNA (part of the cells hnRNA) is colinear with the gene that encodes it, while the mature -globin mRNA is shorter than the gene. The missing RNA was an intron that was removed during RNA processing. 3. Introns are found in most eukaryotic genes that encode proteins, and have also been found in bacteriophage genes.
601 20000 Chapter 5 slide 31
601 20000
Chapter 5 slide 32
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 33
2. Introns typically begin with 5-GU, and end with AG-3, but mRNA splicing signals involve more than just these two small sequences.
a. Spliceosomes are small nuclear ribonucleoprotein particles (snRNPs) associated with pre-mRNAs. b. Spliceosome principal snRNAs are U1, U2, U4, U5, and U6.
3. The steps of splicing are outlined in Figure 5.13: a. U1 snRNP binds the 5 splice junction of the intron, as a result of base-pairing of the U1 snRNA to the intron RNA. b. U2 snRNP binds the branch-point sequence upstream of the 3 splice junction. c. U4/U6 and U5 snRNPs interact, then bind the U1 and U2 snRNPs, creating a loop in the intron. d. U4 snRNP dissociates from the complex, forming the active spliceosome. e. The spliceosome cleaves the intron at the 5 splice junction, freeing it from exon 1. The free 5 end of the intron bonds to a specific nucleotide (usually A) in the branch-point sequence to form an RNA lariat. f. The spliceosome cleaves the intron at the 3 junction, liberating the intron lariat. Exons 1 and 2 are ligated, and the snRNPs are released.
601 20000
Chapter 5 slide 35
601 20000
Chapter 5 slide 36
Self-Splicing of Introns
1. The rRNA genes of most species do not contain introns.
2. Some species of the protozoan Tetrahymena have a 413-bp intron in their 28S rRNA sequence. Tom Cech (1982) showed that splicing of this intron (called a group I intron) is proteinindependent. The intron self-splices by folding into a secondary structure that catalyzes its own excision.
3. Group I introns are rare, but examples occur in: a. Nuclear rRNA genes.
4. The steps in self-splicing of a group I intron in Tetrahymena are shown in Figure 5.14:
a. The pre-rRNA is cleaved at the 5 splice junction and guanosine is added to the 5 end of the intron. b. The intron is cleaved at the 3 splice junction. c. The two exons are joined together.
d. The excised intron forms a lariat structure, which is cleaved to produce a circular RNA and a short linear piece of RNA.
5. Self-splicing is not an enzyme activity, because the RNA is not regenerated in its original form at the end of the reaction. However, the discovery of ribozymes (catalytic RNAs) has significantly altered our view of the biochemistry involved in the origin of life.
601 20000 Chapter 5 slide 38
Fig. 5.14 Self-splicing reaction for the group I intron in Tetrahymena pre-rRNA
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 39
RNA Editing
1. RNA editing adds or deletes nucleotides from a pre-mRNA, or chemically alters the bases, resulting in an mRNA with bases that dont match its DNA coding sequence.
Fig. 5.15 Comparison of the DNA sequences of the cytochrome oxidase subunit III gene in the protozoans Trypanosome brucei (TB), Crithridia fasiculata (Cf), and Leishmania tarentolae (Lt), aligned with the conserved mRNA for Tb
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 41
601 20000
Chapter 5 slide 42
601 20000
Chapter 5 slide 43
Ribosome Structure
1. Ribosomes in both prokaryotes and eukaryotes consist of two subunits of unequal size (large and small), each with at least one rRNA and many ribosomal proteins. 2. E. coli is the model for a prokaryotic ribosome. It is 70S, with 50S and 30S subunits (Figure 5.16).
a. The 50S subunit contains the 23S rRNA (2,904 nt) and 5S rRNA (120 nt), plus 34 different proteins. b. The 30S subunit contains the 16S rRNA (1,542 nt), plus 20 different proteins.
3. Eukaryotic ribosomes are larger and more complex than prokaryotic ones, and vary in size and composition among organisms. Mammalian ribosomes are an example; they are 80S, with 60S and 40S subunits (Figure 5.16).
a. The 60S subunit contains the 28S rRNA (~4,700 nt), the 5.8S rRNA (156 nt) and the 5S rRNA (120 nt), plus about 50 proteins. b. The 40S subunit contains the 18S rRNA (~1,900 nt) plus about 35 proteins. 601 20000 Chapter 5 slide 44
601 20000
Chapter 5 slide 45
Fig. 5.17 Composition of whole ribosomes and of ribosomal subunits in mammalian cells
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 46
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 48
3. Eukaryotes generally have many copies of the rRNA genes (Figure 5.19). a. The three rRNA genes with homology to prokaryotic rRNA genes are 18S-5.8S28S, in that order. In the chromosome these genes are tandemly repeated 100 1,000 times to form rDNA repeat units. The 5S rRNA gene copies are located elsewhere in the genome. b. A nucleolus forms around each rDNA repeat unit, and then they fuse to make one nucleolus. Ribosomal subunits are produced in this structure by addition of the 5S rRNA and ribosomal proteins. c. RNA polymerase I transcribes the rDNA repeat units, producing a pre-rRNA molecule containing the 18S, 5.8S and 28S rRNAs, separated by spacer sequences. i. External transcribed sequences (ETS) located upstream of 18S and downstream of 28S. ii. Internal transcribed spacers (ITS) located on each side of 5.8S. iii. Nontranscribed spacer (NTS) sequence is between each rDNA repeat unit, and includes the promoter. 4. Specific cleavage steps free the rRNAs from their transcript as part of pre-rRNA processing that takes place in the complex of pre-rRNA, 5S rRNA, and ribosomal proteins. The result is formation of 40S and 60S ribosomal subunits, which are then transported to the cytoplasm.
601 20000 Chapter 5 slide 49
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 50
Fig. 5.20 Transfer RNA (a) Cloverleaf structure of yeast alanine tRNA
Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
601 20000
Chapter 5 slide 52
2. Promoter sequences for 5S rRNA and tRNA genes are typically within the sequences that will be transcribed, hence internal control regions (IRC). Promoters for the snRNA genes transcribed by RNA pol III are typically upstream of the genes. 3. Transcription of 5S rDNA produces a mature 5S rRNA, and no sequences need to be removed.
4. Some tRNA genes contain introns. About 10% of the 400 yeast tRNA genes have introns. If present, introns usually are found just 3 to the anticodon, and they are removed by a specific endonuclease, with splicing completed by RNA ligase.
601 20000
Chapter 5 slide 53