Term
| What Catalyses transcription? |
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Definition
| RNA Polymerase (RNAP) + Co-factors |
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Term
| What is the name given to the RNAP + Sigma factor complex? |
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Definition
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Term
| Name the 3 stages of transrcription. |
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Definition
1- Initiation
2- Elongation
3- Termination |
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Term
| What is generated by transcription? |
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Definition
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Term
| How is gene expression regulated in bacteria? |
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Definition
By modifying RNAP:
- Sigma factors alter specificity
- Effector mol. interact w. RNAP
- Promoters can re-direct/re-orient RNAP |
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Term
| Phenotype vs Genotype. Explain |
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Definition
Phenotype is determined by switching genotype ON/OFF.
i.e. genotype is the blueprint which encodes cell hardware. |
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Term
| What can the switch system lead to? |
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Definition
-Infections/immunity
-Survival in extreme cond.
-Tools for genetic manipulation. |
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Term
| Bacteria is used as a simple model system, why? |
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Definition
Some have v. small of genes (~500)
They have well organised genomes
Gene expression is easy to study. |
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Term
| What is the Central Dogma, and who first imposed the idea? |
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Definition
Francis CRICK (1958)
DNA-->RNA-->Protein |
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Term
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Definition
DNA gene----------------->mRNA
^
RNAP holoenzyme
Nucleotide Triphosphates (NTPS)
Mg+ Zn+ |
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Term
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Definition
| Transcribe proteins from RNA |
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Term
| What is the transcription cycle? |
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Definition
| RNAP-sigma + promoter--->RNAPsigma-Pc--->RNAPsigma-Po--->mRNA |
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Term
| What does P-closed and P-open mean? |
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Definition
| It refers to the state of the DNA. |
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Term
| What is the role of the Sigma factor? |
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Definition
It is responsible for recognising + binding to the promoter.
Must leave before transcription can continue. |
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Term
| What are the 7 stages of the transcription cycle? |
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Definition
1-RNAP binds sigma + locates promoter (P-closed)
2-RNAPsigma unwinds DNA (P-open)
3-Transcription begins
4- After ~10nts are transcribed, sigma dissociates and RNAP clamps on
5- Transcription in elongation is processive
6- Termination signals in DNA form RNA structure which destabilises RNAP.
7- Nascennt mRNA is release. RNAP can them bind another sigma. |
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Term
| What are the 4 steps of initiation? |
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Definition
1- Recruitment of sigma + locating promoter.
2- Unwinding of DNA, forming open complex.
3- Synth. of 1st phosphodiester bonds.
4- Ejection of sigma subunit, and RNAP can move past promoter. |
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Term
| What subunits make up the core enzyme? |
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Definition
Alpha
Beta
Beta-Prime
Omega
(with Sigma = Holoenzyme) |
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Term
| Properties of the core Enzyme. |
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Definition
~2000 mol./E. coli
Catalyses RNA synth.
Binds DNA directly
Cannot recognise promoter. |
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Term
| What structure is the RNAP holoenzyme likened to? |
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Definition
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Term
| What are the 2 major Sigma factors? |
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Definition
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Term
| Where does Sigma interact with the promoter? |
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Definition
| At the CONSENSUS SEQ. at postions -35 and -10. |
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Term
| Why is the -10 recognition site a strong promoter region? |
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Definition
| As it is T + A rich, so less H-bonds. Easier to separate DNA. |
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Term
| By what system does Sigma recognise the promoter? |
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Definition
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Term
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Definition
| Direct contact with a.a's + R-groups, giving specificity. |
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Term
| Explain INDIRECT READOUT. |
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Definition
| Enzyme can rwad the outside of the DNA strand (backbone) + indirectly read what is on the inside - backbone is 'lumpy'. |
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Term
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Definition
Regulator of RNAP that mimics an open promoter
non-coding 184nt. molecule.
Prevent transcription from Sigma-70-dependent promoters. |
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Term
| Explain the nt. addition cycle. |
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Definition
-NTPs added @ 3' end of nascent mRNA.
-Catalysis occurs in active centre (i & i+1)
- |
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Term
| What is the co-factor for the nt addition cycle? |
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Definition
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Term
| How does the transition to elongation mode occur? |
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Definition
After ~10nts, Sigma is less tightly bound to RNAP.
its dissociation leads to conformational RNAP change.
Crab-claw now clamps to DNA.
Exit Channel at rear of complex. |
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Term
| What two moving parts are present in the nt addition cycle? |
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Definition
| A Bridge Helix and Trigger Loop/Helix. |
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Term
| What are the two types of termination? |
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Definition
| Intrinsic Termination and Rho-dependent termination. |
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Term
| Explain Intrinsic termination. |
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Definition
A string of AT nt pairs, preceded bya two-fold symmetrical seq.
mRNA dissociates easily (active site is held together by weak U-A bps)
RNA folds into hairpin structure.
Hairpin wedges open RNA. |
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Term
| Explain Rho-Dependent Termination. |
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Definition
Rho protein binds ~40nts behind at RUT region (C-rich) in mRNA
Acts like a 5'-->3' Helicase,
Once Rho reaches transcription site, it dislodges RNAP. |
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Term
| What is anti-termination? |
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Definition
Termination signal are recognised + RNAP is modified to overcome them.
Allows read-though of Rho and intrinsic termination sites. |
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Term
| What is required for anti-termination? |
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Definition
| Encoded accessory pprotein (NusA, B, E, G) |
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Term
| What three ways are there to control gene expression? |
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Definition
1-Epigenetics
2-Control of transcription
3-Post-Translational modifications. |
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Term
| What Sigma factor is prevalent during exponential phase? |
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Definition
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Term
| What sigma factor is prevalent during stationary phase? |
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Definition
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Term
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Definition
613 aa
Growth related genes (~1000) |
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Term
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Definition
330 aa
Stationary phase/stress response genes (~100)
Many genes are under the control of sigma38 |
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Term
| What molecules are +ive and -ive regulators of sigma38? |
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Definition
+ive = ppGpp alarmone (exposure to stress)
-ive = cAMP |
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Term
| What is repsonsible for the rappid turnover of sigma 38 in exponential phase? |
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Definition
ClpP (protease)
Degrades sigma38.
levels of ClpP drops in stationary phase. |
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Term
| What base does sigma38 reccognise, and where is it found? |
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Definition
C @ pos. -13 on the promoter.
(sigma38 is more tolerant of degenerate promoter seq. + spacer length) |
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Term
| What response does ppGpp alarmone co-ordinate? |
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Definition
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Term
| What is the stringent response? |
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Definition
| A response to a.a. starvation |
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Term
| How does ppGpp co-ord. the stringent response? |
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Definition
Directly acts upon RNAP (at initiation stage)
Affects RNAP conformation in open complex
Amplifies differences in promoter. |
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Term
| What does ppGpp stand for? |
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Definition
| Guanosine-tetraphosphate. |
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Term
How does ppGpp exhibit -ive control?
(rRNA genes) |
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Definition
Intrinsic to -ively regulated promoters.
ppGpp interactions with RNAP causes tighter RNAP-DNA innteractions (promoter escape is more difficult).
GC-rich "discriminator" between -10 + start site. |
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Term
How does ppGpp exhibit +ive control?
(a.a. biosynth. genes) |
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Definition
AT-rich "discriminator" between -10 and start site.
ppGpp leads to loosening of RNAP-DNA interactions (escape from promoter is easier) |
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Term
| What two molecules modulate RNAP? |
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Definition
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Term
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Definition
151 a.a protein (small)
Acts synergistically w. ppGpp.
Binds to RNAP NOT DNA.
DksA reduces 1/2lyf of open complex. |
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Term
| What does flipping promoter achieve? |
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Definition
Molecular ON/OFF switch for transcription
Control @ transcriptional level, gives graded response. |
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Term
| What does an "All or None" response allow? |
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Definition
| Allows bacteria to respond to drastically changing environments. |
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Term
| Give an example of flipping promoters. |
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Definition
DIPHASIC SALMONELLAS (1921)
High freq. of ON/OFF switching of genotype. |
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Term
| Name three bacterium display Flipping Promoter level of control. |
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Definition
E. coli - flip promoter and gene B can be transcribed.
Salmonella - Flip promoter and repressor is no longer present to repress gene Z.
Salmonella typhimurium - Two non-allelic flagellin regions (fliC = H2 flagellin/ fljB = H1 flagellin). |
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Term
| What is the biological significance of phase variation? |
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Definition
1-Infection + Immunity
i.e. random switching of flagellin forms
improves chances of causing infection.
2-Environmental Response
Switching in response to temp. (e.coli
fimbriae).
Fimbrial gene expression influenced by
sigma38 |
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