TBR 10 - Expression of Genetic Information Flashcards
Differences between DNA and RNA polymerase.
1) DNA uses dNTPs (releasing PPi) while RNA uses NTPs (also releasing PPi)
2) DNA replication is semiconservative; RNA transcription, DNA eventually rewinds, so it’s conservative
3) DNA requires a primer with a free 3’ OH group to initiate chain elongation. RNA does not require a primer.
4) DNA polymerase almost always involved with synthesizing DNA bidirectionally. DNA directed by RNA polymerase, only one strand of DNA is being copied.
Similarities between DNA and RNA polymerase.
1) Both proceed in the 5’–>3’ direction
2) Both require a DNA bubble for operation.
During DNA replication and transcription, do we see beta and gamma labeled phosphates?
Only in RNA transcription, we would see some beta and gamma phosphates. The first nucleotide has at its 5’ end a beta and gamma phosphate.
Promoter.
RNA polymerase moves along the DNA, looking for proper signals (Ex. TATA box in eukaryotes) that will tell it where to promote transcription. Promoters are upstream from +1 point where transcription begins. Promoter sites bind RNA polymerase.
Is RNA synthesis similar to DNA synthesis.
Aside from the primer, yes. The 3’- OH attacks the alpha phosphate of the next incoming ribonucleotide triphosphate.
Give two kinds of transcription termination/RNA dissociation.
1) Rho independent: in E.coli, the terminator sequence for the end of protein synthesis is a based-paired hairpin sequence on the newly synthesized RNA strand. The hairpin is formed by base pairing of GC on the hairpin structure, following a series of U residues.
2) Rho dependent: rho complex is a protein that has ATPase activity that binds to newly synthesized SS RNA and pulls itself toward the replication bubble where it will dislodge RNA polymerase from the DNA template. `
Three phases of RNA synthesis.
Initiation, elongation, termination.
True or false. All promoters have the same strenght.
False. Promoter strength may vary for particular genes. Ex. Glycolytic genes have strong promoters. Coenzymes, which are present in small amounts for catalytic enzymes have weak promoters.
You will not find the same promoter sequence in front of all genes.
What makes viral promotors unique in terms of weakness of strength?
Viral promoters are particularly strong to attract the RNA of the host cell and force it to transcribe viral RNA polymerase.
Describe the structure of E.coli RNA and the importance of the sigma subunit.
E.coli RNA polymerase is composed of 4 subunits, α2, β, β’ and σ. Together they are called the RNA polymerase holoenzyme. The σ subunit is responsible for locating the promoter site that initiates transcription. Once RNA synthesis begins, the σ will dissociate from the RNA POL.
Apoenzyme.
When a core enzyme (like RNA POL α2, β, β’ ) loses one of its subunits (σ), it is an apoenzyme.
Why is σ factor important for bacterial transcription?
1) lower the affinity of RNA POL for general regions
2) raise binding affinity for one class of promoters
Thus σ is a regulatory subunit that regulates the specificity of RNA POL.
How does RNA POL find its promoter region?
The RNA POL will bind to DNA duplex and then slide along it looking for the -35 and -10 regions. In this search transient, H-bonds are ford with H acceptor and donor groups exposed base pairs. along the grooves of the DNA duplex.
The RNA POL doesn’t repeatedly bind and dissociate.
Summary what goes on in transcription
1) RNA POL binds to the promoter region of the DNA duplex and an INITIATION COMPLEX is formed.
2) As soon as the complex is formed, the σ subunit dissociates.
3) With σ missing, the core enzyme binds more strongly to DNA; DNA template is unwound and read in the 3’ to 5’ direction. This is the transcription bubble.
4) Nascent RNA is synthesized until a termination signal is reached.
During DNA replication, what two processes ensure the accuracy of the new template?
1) editing by DNA polymerase enzymes
2) repair enzymes.
Does RNA polymerase have editing properties? What does this do to the error rate relative to DNA?
No.
10^-10 for DNA versus 10^-4/-5
Enhancer regions of DNA and their typical location.
Enhancers are sequences that also help stimulate transcription. Enhancer regions are usually far away from the promoter region either upstream or downstream. They can even be within the transcribed gene.
Enhancers.
The process of whether or not a gene gets transcribed not only depends on the correct promoter region but also the interaction with specific enhancers. Enhancers are specific proteins that bind to enhancer regions that will allow the RNA POL to sit down on a given promoter region and begin transcription.
Ex. Glucocorticoids can bind to a soluble receptor protein that is specific for a certain enhancer region. Once the hormone receptor complex binds to the enhancer region, transcription begins.
What kinds of modifications can occur on DNA transcripts?
1) trimming and splicing (ex. removing introns)
2) addition of bases to the primary transcript ( like poly A tail).
3) modification of bases; a feature evident in tRNA molecules (ex. uridine to pseudouridine in prokaryotes post transcription).
What happens to the RNA eukaryotic transcript post-transcription ( before translation begins)?
1) capping at the 5 ‘ end; the end is modified with GTP addition. SAM is a methyl donor that adds a methyl group to the N7 position of the guanine. SAM adds methyls tot he 2’ hydroxyls of the adjacent ribose rings, as well. Capping the 5’ end prevents degradation and thereby enhances the stability of the RNA.
2) addition or polyadenine tail at the 3’ end protects from nucleases and phosphatases.
3) splicing which involves excision of introns (intervening sequences). Exons, the genes translated or expressed, are joined together.
What are snRNPs? What is their significance?
snRNPs are RNA molecules that are associated with specific proteins. Small Nuclear Ribonucleoprotein Particles. snRNPS are involved with the base pairing rxns at the spiced junctions of introns and exons.
True or false. Only proteins and their coenzymes can be catalysts.
False. RNA can catalyze reactions by itself. Splicing occurs without enzymes.
Which end are proteins synthesized from?
Proteins are synthesized from the amino end to the carboxyl end (ie the carboxyl end is synthesized last).
What is the direction of translation?
The direction of translation is from the 5’ —> 3’.
