Lecture 16, 17 Flashcards
What is the central dogma?
.
How can cells change or regulate the expression of each gene according to the needs of the moment?
- By controlling the production of mRNA
How does RNA differ from DNA?
- Ribose vs. deoxyribose… so NTPs vs. dNTPs
- Uracil vs. thymine (has methyl)
- RNA is single-stranded
- RNA is very reactive cause exposed bases and 2’OH, while DNA has H at 2’
Although RNA is usually single-stranded, what must it base pair with?
- DNA during transcription
- W/itself to fold into 2ndary structures
Does uracil lacking methyl group seen in thymine affect its Watson-Crick base pairing with adenine?
No
Does RNA form conventional or non-convential base pairing to fold into 3D structures?
Both
What is the fxn of mRNAs?
- coding RNA
- messenger RNA
- code for proteins
What is the fxn of tRNAs?
- transfer RNA
- central to protein synthesis as adaptor btwn mRNA and AAs
What is the fxn of rRNAs?
- ribosomal RNA
- form basic structure of ribosome
- catalyze protein synthesis
What is the fxn of snRNAs?
- small nuclear RNA
- fxn in splicing of mRNA
What is the fxn of snuRNAs?
- small nucleolar RNA
- used to process and chemically modify rRNAs
What is a gene
- the basic physical & functional unit of heredity
- consists of a specific
sequence of nucleotides at a given position on a given chromosome that codes for a specific protein (or, in some cases, an RNA molecule).
Example of a gene?
- trp operon (cluster of genes in prokaryotes)
What does the trp operon code for
- enzymes involved in synthesis of trptophan
During transcription, DNA is used as a ___ to make _____
- Template
- RNA transcript
The ssRNA “transcript”
produced by transcription is complementary to which strand?
And shares the same nucleotide sequence as which strand? With the exception of what?
- template strand
- coding strand, except T’s are U’s now
How does eukaryotic and prokaryotic transcription differ?
- prokaryotic transcription and translation takes place in cytoplasm, COUPLED
- eukaryotic transcription takes place in nucleus while translation in the cytoplasm
- mRNA is modified before leaving nucleus in eukaryotes (5’cap, 3’ polyadenylation, splicing of introns)
In prokaryotes, when is the most critical point in regulating gene expression?
Since translation can start as soon as the end of the 5’ end of the mRNA transcript emerges from the transcription machinery, initiation of transcription is usually the most critical point in regulating gene expression.
In eukaryotes, when is the most critical point in regulating gene expression?
(In eukaryotes, each primary transcript is processed to produce an mRNA, which is
exported out of the nucleus where it is translated - thus each of these steps presents an opportunity to regulate the overall level of gene expression
In bacteria what does mRNA conc depend on?
- the rate of mRNA synthesis
* the rate of mRNA degradation
Explain how mRNA concentration depends on mRNA synthesis and degradation?
Typical life-time of mRNA molecule is minutes or less; this is less time than it takes for cell to divide. Once synthesis of an mRNA stops, the mRNA quickly disappears cause of nuclease degradation, and synthesis of the corresponding protein also stops
3 elements of gene control in prokaryotes in order of importance?
- transcription initiation
- RNA turnover (nuclease degredation)
- transcription termination
What does prokaryotic transcription require (4)
- dsDNA template
- Mg 2+
- RNA pol
- NTPs
How many subunits in RNA pol?
5: alpha, beta, beta’, sigma, w
Fxn of sigma subunit
Promotor recognition (initiation)
Does RNA pol need a primer or helicase during transcription?
No
- RNA polymerase binds tightly to the RNA – does not need a primer
to initiate polymerization – often begins transcription with ATP
-DNA is unwound ahead and rewound behind as RNA is transcribed - RNA pol has helicase activity (does not need a separate helices enzyme)
Where do the NTPs come from for transcription
a channel in the polymerase funnels new NTPs to the polymerase active site
What recognizes the promotor sequences in prokaryotic transcription
sigma70 in RNA pol holoenzyme
How many diff subunits does E.coli have?
7
How is the number scheme on the gene decided?
the DNA base pairs that encode for the RNA are given pos #’s whereas those that represent the region upstream of the coding sequence (regulatory elements) are given neg #’s, counting from the beginning of the coding sequence
- +1 at first nucleotide encoded for RNA, -1 before that… aka no zero
Important sites in promotors in bacteria?
-35 and -10
How are sigma factors numbered
Base on their molecular masses (kDa)
What happens during initiation during prokaryotic transcription (3)
- RNA polymerase holoenzyme forms, binds nonspecifically to the DNA and scans the sequence until it finds a promoter. The RNA pol/DNA complex is in a closed form where the DNA is in a duplex (base-paired) conformation.
- The polymerase partially unwinds the DNA (has helicase
activity) – open complex - and begins transcribing. Synthesis of the RNA transcript begins, with NTPs base-pairing to the template
strand (the sequence matches that of the coding strand, with U
instead of T - After ~10 ribonucleotides are
polymerized the polymerase
undergoes a conformational change, releasing sigma factor and enabling it to move forward rapidly.
What happens during elongation in prokaryotic transcription
Highly processive elongation occurs
What happens during termination in prokaryotic transcription
Newly transcribed RNA only released when a termination signal in DNA encountered
- no new nucleotides are added, the RNA-DNA hybrid dissociates, the
melted region of the DNA rewinds, and RNA polymerase releases the DNA.
Describe the abortion initiation that can occur during initiation?
- Sigma factor interacts very tightly to promotor, preventing preventing pol from translocating very far along the DNA. As synthesis proceeds, DNA becomes compressed w/in polymerase cause pol is bound to promotor but transcribing downstream of promotor, building up stress in the complex
- once 8/9 NTPs polymerized, stress relieved by RNA pol releasing its downstream or upstream contacts. If it releases downstream contacts, RNA transcription aborted. IF it releases upstream contacts, RNA pol escapes the promotor and enter highly proccessive elongation phase
What happens when RNA pol doesn’t have sigma factor
Sigma factor is needed for initiation of transcription, it recognizes specific promotors and gives RNA specificity for DNA of its own species. Without it RNA pol would transcribe any DNA sequence which would be wasteful
What is the rate-limiting step in transcription?
Recognition of promotor
Describe what happens in closed complex vs. open complex in prokaryotic transcription
- During closed complex, RNA polymerase holoenzyme scans DNA and binds tightly to promotor. It is called closed complex cause the DNA has yet to be unwound to expose the template
- During open complex, RNA pol unwinds DNA around -10 region of promotor to form transcription bubble
Which nucleotide does RNA synthesis begin
+1 in 5’ -> 3’ direction
Why is there no primer need for RNA pol?
• aspartates in the RNA pol active site coordinate Mg2+, which binds to the phosphates of the first NTP (usually a purine), presenting its 3’-OH to the next NTP to be added – no primer is required
Is the 1st nucleotide in the RNA polymer (prokaryotes) NTP or NMP. Why?
- NTP, the PPi is not removed
- 5’pppA or 5’pppG
- Since this nucleotide isn’t added to an existing 3’-OH (no primer is required, no nucleophilic attack), inorganic phosphate (PPi, PgPb-) is not released, so the first nucleotide added is a nucleoside triphosphate (NTP).
What is the first nuc in prokaryotic transcription usually
- a purine (A or G)
After the first NTP is added, how is the transcript elongated?
Once the first nucleotide is added, its 3’-OH acts as a
nucleophile, attacking the a-phosphate (Pa) of the incoming NTP (same as in DNA replication).
Does elongation require sigma factor?
No
How is the RNA keep from getting intertwined in the DNA during prokaryotic transcription?
~8 bp of the template strand of the transcription bubble are paired with nascent RNA º ~1 turn of an A-type helix
Can RNA pol start over if it falls off partway
No has to start at a promotor
Why do errors occur much more frequently during transcription than for replication?
RNA polymerases do not have the 3’-5’ nuclease active site found in DNA pol
Why is errors in RNA synthesis not as catastrophic as errors in DNA synthesis?
Since RNA is not the permanent info storage molecule of the cell, errors in RNA synthesis are not passed on to daughter cells and are thus not as catastrophic as errors in DNA synthesis
Even tho RNA pol does not have 3’ -> 5’ nuclease activity, what can it do?
can “backtrack” when a mis-match occurs, recognizing a
distorted RNA:DNA helix and excising it with the help of an elongation factor
What are the two classes of termination signals int he template DNA for prokaryotic transcription
- termination protein, rho (r), a termination
factor - one that is rho-independent
What happens in the rho independent termination?
- have a GC rich sequence in the transcript (so the template strand is also GC rich). The GC rich transcript base pairs with itself to form stable hairpin structure near the end of RNA stand. The hairpin structure destabilizes the RNA:DNA hybrid.
- GC rich template sequence is followed by sequence of 3 or more A’s, which are transcribed into U’s in RNA. This causes weaker H-bonding btwn RNA and DNA hybrid cause only 2-H bonds per A-U. This further destabilizes the RNA/DNA hybrid.
- the combined destabilization induces the RNA to dissociate, terminating transcription
What happens during rho dependent terminators
- a sequence at the end of the gene (termination sequence) causes RNA pol to pause
- upstream of termination sequence the synthesized RNA has 70-100 nucleotide CA-rich sequence called a rut (Rho UTilization)
- hexameric rho protein associates with RNA at the rut and migrates in the 5’ -> 3’ direction, hydrolyzing ATP as it goes, until it reaches transcription complex
- rho disrupts RNA/DNA base pairing and causes release of RNA transcript
Is transcription or DNA replication faster
DNA rep
What polycistronic mRNA
- all genes within an operon are transcribed together onto a single mRNA
- each section/cistron can be translated independently
How are some transcripts made in higher quantities than the others in operons?
Transcription terminators may exist at the ends of the genes
What are housekeeping genes?
- constitutively expressed
- code for enzymes required by cell at all times
What are DNA binding proteins
- activator or repressors
What are effectors
Bind to DNA binding proteins (activator or repressors) and alter their affinity for DNA
What does it mean to be inducible?
Gene products that inc in conc under a given condition
What does it mean to be repressible?
Gene products that dec in conc under a given condition
What is induction and repression of a gene controlled by?
- controlling RNA pol interactions with promotors
- control most often affected by modulating promotor activity via protein binding
- regulatory proteins bind at or “near” promoter
What is negative regulation?
A repressor protein binds to the DNA at a site at or near the promotor called the operator, and blocks RNA pol activity during transcription
What is positive regulation
Counters neg regulation, an acitvator binds to DNA & enhances activity of RNA pol during transcription
The affinity of both repressors and activators can be modulated by binding of \_\_\_ molecules.
Effector
How does effectors binding to repressors affect transcription?
Effectors can bind to repressor to increase or decrease their affinity for the operator… two types of effectors for repressors are:
- inducers: decrease affinity of repressor for operator
co-repressors: inc affinity of repressor for operator
How does effectors binding to activators affect transcription?
Effectors can bind to activators to increase or decrease their affinity for the operator
What are repressors encoded by
Regulatory genes located close to genes whose expression they repress
What does the operon consist of
Structural genes, promotor, operator
- not regulatory gene itself
Example of activator that binds to DNA only after binding to an activator?
- activator CAP binds to effector cAMP then binds to DNA near lac promotor and recruits RNA pol
Example of effector that binds to repressor to decrease its affinity to the operator? (aka an inducer)
- Allolactose binding to the lac repressor
Example of effector that binds to repressor to increase its affinity to the operator? (aka an co-repressor)
- Tryptophan binding to trp repressor
An example of both negative and pos regulation in bacteria?
Lac operon
How is lactose hydrolyzed into glucose?
Using enzyme b-galactosidase
Three structural genes on lac operon and what they code for?
- Gene Z: b-galactosidase; hydrolyzes lactose to glucose
- Gene Y: Galactoside permease; transport lactose across bacterial membrane
- gene A: Thiogalactoside transacetylase; export unwanted toxins that enter cell due to permease activity
What happens during high levels of glucose and low lactose
Lac operon repressed, glucose expressed
What happens during high levels of lactose and low glucose
Lac operon expressed, glucose repressed
What does lac I gene code for?
Lac repressor protein or Lac I
What does the lac operator overlap with
lac promotor
How is the lac operon negatively regulated?
In absence of lactose, lac operon is repressed with Lac repressor bound to operator
When lactose is present, how is it converted to glucose/galactose?
By existing b-galactosidase in the cell
Besides hydrolyzing lactose to glucose/galactose what else does b-galactosidase do?
Isomerize some lactose to allolactose
What is an inducer not hydrolyzed by b-galactosidase?
IPTG
How is IPTG used to experimentally induce gene expression?
The lac promoter (P) and operator (O)
are inserted into the plasmid and the gene of interest is placed after the lac promoter/operator
(in the MCS) so that
its expression is under control of that promoter. Expression of the gene can be induced by IPTG.
Lac repressor binds DNA via what?
Helix-turn-helix motif
What is a helix-turn-helix motif
- common motif in DNA binding proteins
- 2 alpha-helices joined by tight turn
What is the role of each alpha helix in a helix-turn-helix motif
- 2nd helix aka recognition helix lies in DNA major groove where side chains contact the bases
- 1st helix interacts w/sugar-phosphate backbone
What are regulatory proteins that bind DNA via the helix-turn-helix motif as dimers?
- Lac repressor
- CAP
- Trp repressor
How is lac operon positively regulated?
- By CAP in the presence of cAMP
- CAP binds to cAMP which changes its conformation and inc its affinity for lac promotor
How does CAP-cAMP binding stimulate transcription?
- Distorts DNA, allowing RNA pol to bind more effectively, or by binding directly to RNA pol
Why is CAP not an effective activator in high levels of glucose?
In high lvls of glucose, adenylate cyclase is inhibited so cAMP lvls are low and CAP is not a effective activator.
- known as catabolite repression cause a catabolite of glucose reduces (cAMP)
What happens when there’s no lactose present
- lac operon switched off (repressed)
* essentially no lac mRNA synthesized regardless of [glucose]
What happens when there’s lactose present and no glucose
the inducer allolactose is produced, binds to the lac repressor and inactivates it
• no glucose is present so [cAMP] is high; cAMP binds CRP, which can then bind the lac
promoter and stimulate transcription
• high level of transcription
What happens when lactose and glucose is present
• the inducer allolactose is produced, binds to the Lac
repressor and inactivates it
• glucose present so [cAMP] is low and thus CRP does not ‘help’ transcription - catabolite repression
• only a low level of
transcription occurs
