Gene Expression 10. 19.12 Flashcards
Learning Objectives
- Compare and contrast the differences between DNA and RNA stucture
- Explain why the 2’-hyroxyl group of RNA allows it, and not DNA, to readily fold
- List and describe the major classes of RNA, both coding and noncoding
- Explain how transcriptional initiation, elongation, and termination work and how RNA polymerase mediates each step
- Compare and contrast the differneces between bacterial and eukaryotic transcription
- Explaiin why supercoiling occurs during transcription and identify the enzymes that resolve supercoiling
- Describe the clinical relevance and basis of action of actinomycin D, alpha-amanitin, rifampin
Does transcription and translatino require more steps in eukaryotes or bacteria
Eukaryotes
what can occur with teh additional steps for eukaryotes?
provides euk with sites for regulatino but also create opprotunites for htings to go wrong and cause disease
what can an overexpression of oncogenes lead to?
Cancer
Why do physicians need to study bacteria? How much more bacteria are present on our body than human cells?
10x more bacterial cells
What is the target and mechanism of RIFAMPIN
To treat Tuberculosis
What is the target and mechanism of active agent in poisonous muchrooms
alpha amantin
What is the target and mechanism of the antibiotic Actinomycin D
used to ttreat some pediatric cancers
What are the ajor tyeps of RNA (6)
- mRNA
- rRNA
- tRNA
- miRNA
- siRNA
What is mRNA
messenger RNAs,
Code for proteins
For Eukaryotes, genes are transcribed as precursors, or pre-mRNAs
These precursors are modified iwth at CAP on their 5’ end and a poly(A)tail on their 3’ end
How are mRNAs modified in Euk?
the are modified with a CAp on their 5’end and a poly (A) tail on their 3’ end.
How are they processed further? (mRNA)
they must be furthe rocessed by splicing
mRNA is designated a coding RNA, whereas the following are designated noncoding RNAs
What are rRNAs
ribosomal RNAs, form the basic structure of the ribosome and catalyze protein synthesis
How many differenr rRNAs make up the ribosomes?
3-4 different rRNAs and several dozen robosomal proteins
What are transfer RNAS 9tRNA)
central to protein synthesis as ADAPTORS between mRNA and amino acids
they are adapotr moleucles in translation
what are miRNA
Micro RNA- regualte gene expression typically by blocking translation of selective mRNAs
how is miRNA transcribed?
like mRNA, transcribed as a precursor
What does miRNA regulate after processing?
miRNAs regulate either transcription or translation
what are siRNA
small interfering RNA- turn off gene expression by directing degradation of selective mNAs and the establishment of compact chromatin structures
siRNA destroys foreign RNA and may have improtnat therapeutic applications
What are other noncoding RNAs
function in diverse cell procesess, including telomere syntehsis, X-chromosome inactivation, and teh transport of proteins into the ER
many functions are unknown
What type of RNAs are disgnted as coding RNA?
mRNA
What process of protein synthesis is tRNAs invovled in (transcription or translation)
TRANSLATION
what are snRNAS
small nuclear RNAs,
Funciton in a variety of nucealr processes, including splicing of pre-mRNA
what are snoRNAs
small nucleolus RNAs, used to process and chemically modify rRNAs
What are siRNAs
small interfering RNAS
How do siRNAs regulate gene expression?
Direct degradation of selective mRNAs and establish compact chromatin structures
Whatdo siRNAs do to foreign RNA?
Destroys foreign RNA and may have important therapeutic applciations
What do other noncoding RNAs do?
function in diverse cell processes
Telomere sntehsis
X-chroosome inactivation
Transport of proteins into the ER
Which Carbon on the ribose sugar does the base attach to
C #1
What are the two chemical differences between DNA and RNA?
RNA has a 2-OH group instead of the 2’-H atom of DNA
- RNA uses uracil (U) instead of thymine (T)
How does the 2’ OH atom of RNA alter teh properies of the polymer (in comparison to DNA)
3 ways
- 2’OH causes RNA duplexes to form A-form helices, not B-for helices as in DNA
- it allows RNA to fold
- it makes RNA significantly more susceptible to hydrolysis
How can RNA fold while it is single stranded?
B/c it is stabilized by its 2’OH gorups
What is the most common hsape an RNA can it fold into?
it can fold into complex shapes but the most common structural element in folded RNA is the DOULBE STRANDED A-FORM HELIX
Which helix form is DNA? RNA? (alpha or beta)
DNA- beta form
RNA-alpha form
Is the major or minor groove more accessible in RNA helix?
In alpha helix (RNA_< major group is narrow and shallower…the Minor groove (m) is now large and most accessible surface feature of A-form helix
minor groove is more accessbile in RNA
Which groove presents unique H-bonding patterns to permit site-specific recognition by proteins?
Major groove
Why is there limited recognition opportuniteis in teh minor groove of RNA
the H-bond pattern in this groove cannot be used by proteins to distriminate between A, C, G, and U
What kind of H bonding can DNA phosphodiester backbone participate in?
only H-bond acceptors
What kind of phosphodiester backbone does RNA have?
the 2’OH group of RNA contains both H-bond donors and acceptors
Where do the H bonds for RNA come from?
There are 2 lone pairs of oxygen atom offer 2 H-bond acceptors
1 H atom provides 1 H-bond donor
Ability to donate and accept H-bonds does what?
Ability to donate and accept H-bonds allows RNA to stabilize long range interactions adn thus fold to create active sites that catalyze reactions like peptide bone formation
What does the base triple between one nucleotide form tRNA , anotehr from mRNA and a thrid from the ribosiome demonstarte?
the stabilizing role of 2’ OH groups
These 2’ OH interactions shown occur in all ribosomes and are essential for mRNA decoding
What kind of interactions allow ssRNA to fold???
Non-Watson-Crick interactions and 2’OHs
What kind of secondary structures does RNA fold into? Tertieary?
Helices, stem loops, and juncitons. These secondary structural elements then come togther to create a tertiary folded structure
What is the most common structure in folded RNA?
What kind of pairs is this?
A-form helix
Only Watson-Crick bp fold into a classic A-form helix
What do various combinations of non-Watson-Crick pairs create ?
distortions or bulges in teh helices and many other wondrous motifs thata re building blocks of the folded structure
base triples and even base quadruples
What is a wobble pair?
Common no-Watson-Crick bp is a GU wobble pair
All possible cobinations ar foudn in these non-A-form RNA structures
What stabilizes the non-Watson-Crick basepairs?
interactions with 2’ OH group
What does gene expression begin with?
Trascription, which copies DNA into RNA
What kind of RNA is made with transcription?
RNA is either coding RNA (mRNA) or noncoding RNA
mRNA is translated into protein by the ribosome whereas noncoding RNAs are end products like tRNA or rRNA
How are eukaryotic RNA different from bacteria mRNA
Euk messages are transcribed as a precursor mRNA (pre-mRNA)
What modifications must be done to the pre-mRNA in order to produce the mature mRNA
pre-mRNA must be modified by 5’ capping, splicing and 3’-polyadenylation
What is RNA transcription catalyzed by?
template driven polymerase with nucleotide triphospahte NTP substartes
How does RNA Transcription contrast with DNA replication (5 ways):
- It produces ss product instead of ds one
- It doesn’t require a primer
- It uses NTP (ribonucleotide substrate) not dNTPs (deoxynucleotide substrates);
- it uses U bases instead of thyine ones
- It has lower fidelity! DNA polymerases have editing sites and as a result they are highly accurate, making about 1 mistake/10 million nucleotides
RNA Polymerase in contrast, do not have editing sites and as a result are not asaccurate, making 1 mistake/10,000 nucleotides
what three steps do RNA polymerases carry out?
Initiation, Elongation, Termination
Describe how difficult initiation of transcription might be?
Starts by lcoating correct start site
This is challengin in bacteria (E. coli), b/c there could be over 4.6 millino potential start sites
IT is more difficult in humans wher ethere could be 3.2 billion potential start sites
Ds DNA must be melted to expose the template strand for copying –> transcription bubbl
What is a transcription bubble?
it is a transietn stie made by melting of the strands, to expose template strand for copying
What occurs during Elongation step?
Stepwise and iterative process that catalyzes teh addition of one nucleotide at a time until the termination signal occurs
At end of each elongation step a 3’ hydroxyl group is exposed on the growing RNA polymer
What happens at the end of each elongation step?
3’ OH group is exposed on the growing RNA polymer
Only when the base of the incoming NTP forms a Watson base pair (WC bp formation is requried) with teh base of the template strand located in the active site is this 3’OH group activated to attack the alph phosphate group (phaopste closest t sugar moetiy) of this incoming NTP
This reaction releases pyrophosphate (PPi) to drive reaction forward
What is termination? What is the product?
process of stpping transcripton at the correct site. The resultant product is a single stranded RNA, which often folds
Why is Polymerase Orientation important during transcription?
Oritentation of Poly defines which starnd is the template
What provides the directions for intiiation and termination >
DNA sequence offers a road map of sorts (promotoer and termiantion sites.
What is the signifiance of sites at -10 and -35 (assymmettry_
critical to define directino in which polymerase binds to template
How do proteins recognize these different sequences that direct initiation and terminatino?
Proteins dock into teh acessible major groove of the B-form DNA helix
reads out sequence by forming complementary interactions iwth accessible functinoal groups at the adges of the bases
What does the red shaded region lead to (pg 14)
a sequence that will form a stem loop structure that signals termination.
What is the sigma factor? Function? (bacterial RNA pol)
A subunit of the RNA polymerase holoenzyme, that recognizes the asymmetric promotor sequence located just upstream of the start nucleotide
What composes the holocoplex? (bac RNA pol)
DNA promotor, sigma factor, and RNA Polymerase
What ocurs with the assembly of the holocomplex? (Bac RNA Pol)
ATP independent helix unwinding to expose bottom template strand for transcription
How many nucletotides are transcribed (RNA pol bac) before sigma factor interaction with the promotor weaken
about 10
sigma factor interactions with the promotor weaken
How does the RNA Pol (bacteria) transition form intiiation mode to elongation mode?
Conformation change in teh polymerase allows it to wrap itself aroudn the incoming ds DNA, making this enzye more processive (favoring continuing Tx rather thtan falling off the template, which would prematurely terminate transcription)
What happens to sigma and RNA poly (bac) during elongation?
polymerase is highly processive and the sigma factor dissociates
What are the two mechanisms for termination of RNA Pol (bact)
. stem-loop structure, which leads to release
- other option employs the p (Rho) protein, which is a helicase that dissociates the RNA transcript form teh DNA tempalte
What does termination involve?
release of transcribed ss RNA product and release of ds DNA
What is supercoiling?
coiling of DNA that causes HELICAL TENSION
In order to create the transcription bubble…
DNA must rotate to alleviate the srai caused by unwinding
What are positive supercoils and negative supercoils
Positive supercoils precede and negative supercoils follow the action of RNA polymerases
Positive superocils
upstream of RNA polymerase
+ superhelical tension ahead of polymearse makes DNA helix more difficult to open p (unwind to create a bubble) but helps unwrap DNA helix from teh nucleosome
What removes superhlical tension in euk?
Topoisomerases
What do bacterail cells do with suprcoils?
bacteria have specialized ATPase called DNA gyrase that uses energy of ATP hyrdolysis to pump supercoils into DNA
DNA gyrase will either increse or decrease helical tensino so that bacterial chromosome maintains a constant negaative supercoil
What does negative supercoil do? (bactera)
makes it easer to unwind DNA , thereby adiing bacterial transcription
How many RNA polymerases do baceteria have?
one
How many RNA polymearses do eukaryotes have?
three main RNA polymerases
What are the three RNA Polymerases
RNA Pol I- transcibe rRNAs along with a few otehr small RNAs
RNA Poly II- transcribes all the coding RNAs (the mones that become mRNAS as well as some nonconding RNAs)
RNA Poly III- transribe rRNAs along with a few other small RNAs
What does poisonous mushroom amanita phalloides preferentially inhibit?
RNA Pol II
shutting down all protein production
What genes do RNA Polymerase I transcribe?
5.8S, 18S, 28S rRNA genes,
insensitive to Amantin
RNA Poly II transcribes?
all protein-coding genes, plus snoRNAs genes, miRNA genes, siRNA genes, and moost snRNA genes
Stronngly inhibited by amantin
RNA pol II transribes?
tRNA genes, 5S rRNA genes, some snRNA genes and genes for other small RNAs
Only inhibited at high concentrations
what does “S” value mean?
refer to reate of sedimentation in an ultracentrifgue. The larger the S value, the larger the rRNA
Which polymerases are used to make Eukaryotic Ribosome Biogenesis? (eRB)
RNA Pol I, II, and III
What does Pol I in eRB
Pol I transcribes the pre-rRNA that embeds 3 or f the four rRNAs of the ribosome
RNA pol II transribes?
tRNA genes, 5S rRNA genes, some snRNA genes and genes for other small RNAs
Only inhibited at high concentrations
what does “S” value mean?
refer to reate of sedimentation in an ultracentrifgue. The larger the S value, the larger the rRNA
Which polymerases are used to make Eukaryotic Ribosome Biogenesis? (eRB)
RNA Pol I, II, and III
What does Pol I in eRB
Pol I transcribes the pre-rRNA that embeds 3 or f the four rRNAs of the ribosome
What is the purpose
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