Exam 3: Chp 26 and Partial 27 Flashcards
What is Transcription?
Transcription is the first step in gene expression, in which information from a gene is used to construct a functional product such as a protein. The goal of transcription is to make a RNA copy of a gene’s DNA sequence. For a protein-coding gene, the RNA copy, or transcript, carries the information needed to build a polypeptide (protein or protein subunit). Eukaryotic transcripts need to go through some processing steps before translation into proteins.
The main enzyme involved in transcription is RNA polymerase, which uses a single-stranded DNA template to synthesize a complementary strand of RNA. Specifically, RNA polymerase builds an RNA strand in the 5’ to 3’ direction, adding each new nucleotide to the 3’ end of the strand.
Transcription has three stages: initiation, elongation, and termination.
DNA contains the information needed to make all of our proteins, and that RNA is a messenger that carries this information to the ribosomes (RNA–protein complexes)
This copy, called messenger RNA (mRNA), carries the gene’s protein information encoded in DNA. In humans and other complex organisms, mRNA moves from the cell nucleus to the cell cytoplasm (watery interior), where it is used for synthesizing the encoded protein.
How does RNAP recognize the correct DNA strand and initiate RNA synthesis at the beginning of a gene (or operon)?
RNAP binds to its initiation sites through base sequences known as promoters that are recognized by the corresponding σ70 factor.
The core enzyme initiates transcription only at promoter because of the σ (σ70 is predominant in E. coli).
the σ subunit does not stay closely associated with the core enzyme (αββ’ω) except when helping to initiate transcription.
Once RNA polymerases are in the right place to start copying DNA, they just begin making RNA by joining together RNA nucleotides complementary to the DNA template.
Initiation of transcription requires binding of RNA polymerase to a small single strand portion in the promoter of a gene.
RNA synthesis is normally initiated at specific sites on the DNA template.
What is a promoter? Where is the “+1 position” in a promoter?
A promoter is a non-coding sequence and a region of DNA upstream of a gene where relevant proteins (such as RNA polymerase and transcription factors) bind to initiate transcription of that gene. The resulting transcription produces an RNA molecule (such as mRNA)
The holoenzyme forms tight complexes with promoters.
In prokaryotes, the promoter consists of two short sequences at -10 and -35 positions upstream from the transcription start site.
+1 position is the position where the first nucleotide is synthesized during transcription.
A base pair in a promoter region is assigned a negative or positive number that indicates its position, upstream or downstream in the direction of RNAP travel, from the first nucleotide that is transcribed to RNA; this start site is +1 and there is no 0. Because RNA is synthesized in the 5′ → 3′ direction (see below), the promoter is said to lie upstream of the RNA’s starting nucleotide.
upstream DNA is the DNA which occurs towards the 5’ end from a particular point on the DNA strand this DNA mainly contain the regulatory elements of a gene, including the promoter site and the transcription binding sites whereas the downstream DNA is the DNA which occurs towards the 3’ end The downstream DNA of a gene contains the protein-coding region of the gene. In eukaryotes, it contains exons and introns. The protein-coding region undergoes transcription to produce a functional RNA molecule
The purpose of the promoter is to bind transcription factors that control the initiation of transcription.
What are the consensus sequences in the -10 region—Pribnow box and -35 region in E. coli?
In prokaryotes, the promoter consists of two short sequences at -10 and -35 positions upstream from the transcription start site.
The sequence at -10 is called the Pribnow box, or the -10 element, and usually consists of the six nucleotides TATAAT. The Pribnow box is absolutely essential to start transcription in prokaryotes.
The other sequence at -35 (the -35 element) usually consists of the six nucleotides TTGACA. Its presence allows a very high transcription rate.
To get a better sense of how a promoter works, let’s look an example from bacteria. A typical bacterial promoter contains two important DNA sequences, the - 10 and -35 elements.
RNA polymerase recognizes and binds directly to these sequences.
Once the RNA polymerase has bound, it can open up the DNA and get to work. DNA opening occurs at the -10 element, where the strands are easy to separate due to the many As and Ts (which bind to each other using just two hydrogen bonds, rather than the three hydrogen bonds of Gs and Cs).
They come 35 and 10 nucleotides before the initiation site. The minus signs just mean that they are before, not after, the initiation site.
The footprint of RNAP holoenzyme indicates that it contacts the promoter primarily at its –10 and –35 regions.
which regions of the σ factor bind to the -10 and -35 regions, respectively?
- σ70 factor can be divided into four regions: region 1, 2, 3 and 4.
- Region 4 possesses a common DNA binding motif called a helix-turn-helix. Region 4 binds to the -35 element through this motif. One of these helices inserts into the major groove and interacts with bases in -35; the other lies on the top of the groove and contacts with the backbone.
- Region 2 forms a α helix that binds to -10 region where the DNA melting is initiated during the transition from the closed to open complex. Thus, the region 2 does more than simply DNA binding—DNA melting, ssDNA stabilization.
- Region 3 interacts with extended -10 where present.
From which nucleotide is transcription mostly initiated?
The initiating (+1) nucleotide is mostly A or G.
How many strands of DNA are copied in transcription? What is the template and non template strand?
1,In contrast to replication, which requires that both strands of the chromosome be entirely copied, the regulated expression of genetic information involves the copying of much smaller, single-strand portions of the genome.
The DNA strand that serves as a template during transcription is known as the antisense or noncoding strand because its sequence is complementary to that of the RNA. The other DNA strand, the nontemplate strand, which has the same sequence as the transcribed RNA (except for the replacement of U with T), is known as the sense or coding strand.
Most eukaryotic genes are transcribed individually. However, prokaryotic genes are frequently transcribed together. These genetic units are called?
operons, typically contain genes with related functions
Overview of initiation using terms, closed and open complex and bubbles
RNA polymerase, together with initiation factors in many cases, binds to a specific region of the promoter of a gene (-10 and -35 region in E. coli), forming a close complex. The DNA around the point where transcription will start unwinds and the base pairs are melted, producing a “bubble” of a single-stranded DNA and forming a open complex. DNA melting occurs between positions -9 and +2 relative to the transcription start site.
• Unlike DNA replication, transcription is initiated by RNA polymerase without the need for a primer.
How do alternative σ factors direct RNA polymerase to alternative promoters?
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Gene Expression Is Controlled by Different σ Factors:
In bacteriophage: The bacterial phage uses three sigma factors in succession to regulate expression of its genome. This ensures that viral genes are expressed in the order in which they are needed.
In E. coli: heat shock induces the amount of a new sigma factor, sigma 32, which displaces sigma 70 form a proportion of RNA ploymerases, and direct those enzymes to transcribe genes whose products protect the cell from the effects of heat shock
In what direction does the RNA chain grow?
The RNA Chain Grows from the 5′ to 3′ End
which regions of the σ factor bind to the -10 and -35 regions, respectively?
σ Factor Mediates Binding of Polymerase to the Promoter:
- σ70 factor can be divided into four regions: region 1, 2, 3 and 4.
- Region 4 possesses a common DNA binding motif called a helix-turn-helix. Region 4 binds to the -35 element through this motif. One of these helices inserts into the major groove and interacts with bases in -35; the other lies on the top of the groove and contacts with the backbone. * Region 2 forms a α helix that binds to -10 region where the DNA melting is initiated during the transition from the closed to open complex. Thus, the region 2 does more than simply DNA binding—DNA melting, ssDNA stabilization.
- Region 3 interacts with extended -10 where present.
What happens before entering the elongation phase for E.coli?
When does regulation of gene expression occur?
RNA polymerase synthesizes several short RNAs before entering the elongation phase caused by Strain that builds up in the DNA template. This period is called abortive initiation.
releases after only ∼9 to 11 nt have been polymerized,
The regulation of gene expression generally occurs in the step of transcription initiation.
In abortive initiation, the RNAP fails to escape the promoter and instead relieves the conformational tension by releasing the newly synthesized RNA fragment, thereby letting the transcription bubble relax to its normal size. The RNAP then reinitiates transcription from the +1 position.
Close complex and open complex
RNA polymerase, together with initiation factors in many cases, binds to a specific region of the promoter of a gene (-10 and -35 region in E. coli), forming a close complex.
The DNA around the point where transcription will start unwinds and the base pairs are melted, producing a “bubble” of a single-stranded DNA and forming a open complex. DNA melting occurs between positions -9 and +2 relative to the transcription start site.
Describe Elongation in Transcription in prokaryotes
- Elongation: once the RNA polymerase has synthesized a short stretch of RNA (~10 bases), the transcription shifts into elongation phase. This transition requires further conformational changes in polymerases that lead it to grip template more firmly.
- RNA polymerase is processive during chain elongation.
- Like replication, transcription always occurs in a 5’ to 3’ direction. i.e. the ribonucleotide is added to the 3’ end of the growing chain.
- Transcription is rapid. The in vivo rate of transcription is 20 to 50 nucleotides per second at 37oC.
- During elongation, the enzyme complex performs additional tasks besides catalysis of RNA synthesis.
What are the functions of RNA polymerase besides catalyzing RNA synthesis?
- During elongation, the enzyme complex performs additional tasks besides catalysis of RNA synthesis including unwinding the DNA in front and re-annealling it behind, dissociating the growing RNA chain from the template as it moves along, and proofreading the synthesized RNA.
What is the significance of RNA association with the template only in few base pairs?
• Only a few bases are paired between RNA and its template DNA.
• The displacement is critical for the RNA to be translated to produce its protein products in prokaryotes.
• This association ensures the gene can be transcribed by multiple RNA polymerase at the same time. Thus, a cell can synthesize large number of transcripts from single gene in a short time.
What is it indicated that a actively transcribing gene shows a arrowhead-like shape under electron microscope?
The synthesis of RNAs that are needed in large quantities, rRNAs, for example, is initiated as often as is sterically possible, about once per second. This gives rise to an arrowhead appearance of the transcribed DNA
The “arrowhead” structures result from the increasing lengths of the nascent RNA chains as the RNA polymerases synthesizing them move from the initiation site on the DNA to the termination site.
Electron micrograph indicates that DNA contains specific sites at which transcription is terminated.
Where does transcription stop in prokaryotes?
At specific sites which require Rho factors to terminate transcription
once the polymerase has transcribed the length of the gene, it must stop and release the RNA product. This process requires transcription termination sequences—the terminators and often proteins binding to the terminators. Some terminators are wellcharacterized but others are less clear.
What is termination in transcription in prokaryotes?
Termination: once the polymerase has transcribed the length of the gene, it must stop and release the RNA product. This process requires transcription termination sequences—the terminators and often proteins binding to the terminators. Some terminators are wellcharacterized but others are less clear.
What is a terminator and what are the two types?
- Terminator: a specific sequence downstream of the gene coding region functioning for transcription termination.
- Two types of terminator: Rho-independent and Rho-dependent.
Mechanisms of Rho-independent and Rho-dependent terminators.
Transcription Termination by Rho-independent Terminator—
* The hairpin forms in the RNA as soon as this region has been transcribed.
- The hairpin structure disrupts RNA polymerase just as it is transcribing the AT rich stretch.
- The combination of the hairpin structure and the weak interaction between Us and As conspires to dissociate RNA from the DNA template.
Rho-dependent Terminator—
* Rho-dependent terminators are less well-characterized.
- Rho (ρ) is a ring-shaped protein with six identical subunits, binds to single-stranded RNA when RNA exits from the polymerase.
- Rho is directed to a RNA by binding to the so-called rut (for Rho utilization) site consisting of 80-100 nucleotides that do not fold into a secondary structure and are rich in C residues but poor in G.
- Rho only binds to those transcripts still being transcribed beyond the end of a gene, but not the transcripts being translated.
- Rho is a helicase that unwinds RNA-DNA and RNA-RNA double helices by translocating along a single strand of RNA in 5’-3’ direction. This process uses the energy derived from NTP hydrolysis activity from Rho.
Difference between prokaryotes and eukaryotes transcription polymerases?
eukaryotic transcription is distinguished by having 3 RNAPs and by much more complicated control sequences. prokaryotes only have 1
Bacteria require only one additional initiation factor (σ), whereas several initiation factors, called the general transcription factors (GTFs), are required for efficient and promoter-specific initiation in eukaryotes
Pol I, II, and III are responsible for the synthesis of what different types of RNA?
- RNA polymerase I (RNAP I), which is located in the nucleoli and synthesizes the precursors of most rRNAs.
- RNA polymerase II (RNAP II), which occurs in the nucleoplasm, synthesizes the mRNA precursors.
- RNA polymerase III (RNAP III), which also occurs in the nucleoplasm, synthesizes the precursors of 5S rRNA, the tRNAs, and a variety of other small nuclear and cytosolic RNAs.
- In addition, eukaryotic cells contain separated mitochondrial and (in plants) chloroplast RNA polymerases responsible for transcription of genes in these organelles.
Mechanisms of rifampicin, actinomycin D, and amatoxin to inhibit transcription.
rifamycin B,- specifically inhibit transcription by prokaryotic but not eukaryotic RNA polymerases. The selectivity and high potency of rifampicin (2 × 10 −8 M results in 50% inhibition of bacterial RNA polymerase) make it a medically useful bactericidal agent. Rifamycins inhibit neither the binding of RNA polymerase to the promoter nor the formation of the first phosphodiester bonds, but they prevent further chain elongation. The inactivated RNA polymerase remains bound to the promoter, thereby blocking initiation by uninhibited enzyme. Once RNA chain elongation has occurred, however, rifamycins have no effect on the subsequent elongation process.
Actinomycin D (right), a useful antineoplastic (anticancer) agent produced by Streptomyces antibioticus, tightly binds to duplex DNA
and, in doing so, strongly inhibits both transcription and DNA replication, presumably by interfering with the passage of RNA and DNA polymerases.
The poisonous mushroom Amanita phalloides (death cap), which is responsible for the majority of fatal mushroom poisonings in Europe, contains several types of toxic substances, including a series of unusual bicyclic octapeptides known as amatoxins. which is representative of the amatoxins, forms a tight 1:1 complex with RNAP II (K = 10 −8 M) and a looser one with RNAP III (K = 10 −6 M) so as to specifically block their elongation steps.
