Chromatin and Transcription Initiation and elongation Flashcards
Histone Acetylation
- Importance of post-translational modifications of the histone tails.
- Changes in charge (Acetyl group is negatively charged) will promote nucleosome dissociation. It also may change the potential protein partner that could bind to the histone tails.
- The acetylation is reversible (HAT vs. HDAC). At least 10 different HDACs have been identified. HAT exists in two forms HAT-A (nuclear) and HAT-B (cytoplasmic)
N-terminal tail
histone-fold domain
Lysine modified by addition of acetyl group (HAT) or removal (HDAC)
Histone Acetylation
HAT can form a complex with an activator
HDAC (1 to 12) can form a complex with a repressor
Coactivator (HAT)+ Activator + acetyl group =Acetylated histones-active chromatin
Co-repressor(HDAC) + repressor=deacetu;ate histones - inactive chromatin
Hat is a co-activator (often needs to be brought in contact with histone tails). HDAC is a co-repressor requiring a partner to find histone tails
histone methylation and phosphorylation
Active chromatin - H3 N-terminal tail
Inactive chromatin
the histone N-termini are the target of the modification
Multiple methyl groups can be present (tri-methylation of Lys 4,9 or histone H3, Lys 20 on H4, play a role in regulation
Other histon modifications: Phosphorylation and methylation. Different charges and conformations will dictate the function
Summary of location and type of histone tail modifications
What is the role of histone in post-translational modifications
- Repression/silencing
- Inprinting (silent allele)
- Activation
Sumo: Small Ubiquitin-like modifier 18% identity with Ub, similar 3D structure
TSS: transcription start site
Nucleosome remodeling factors
Binding of transcriptional activatio and chromatin remodeling faction
Chromatin remodeling factor + activator
Nucleosome displacement
Binding of general transcription factors and RNA polymerase
What is the role of nucleosome remodeling complexes
sliding, changes in DNA path partial dissociation
ATP dependence (through SWI2 or ISWI sub-unit)
a. translocation-dependent remodeling = SWI/SNF and ATP is converted to ADP considered Nucleosome sliding
b. remodeling of DNA contacts
ATP to ADP SWI/SNF Changes in DNA path
c. remodeling of octamer structure
Loss of H2A-H2B dimer
Swi/Snf, NURF, CHRAC, NURD
All complexes are ATP-dependent
Epigenetic inheritance of histone modifications
- Parental chromatin
- DNA replication
- Parental nucleosomes distributed to progeny strands
- Incorporation of new nucleosomes
- Parental modified histones direct modification of newly incorporated histones
Regulation of transcription by siRNAs
- siRNA-association with RITS unwinding of siRNA
- Pairing with mRNA transcript at target gene
- mRNA + siRNA + RITS
- methylation of H3 lysine-9 heterochromatin formation
- transcription repressed
RITS: RNA-induced initiation and transcriptional Gene silencing
Chromatin Structure and Enhancers
Role of nucleosomes as a structural element that will redirect the DNA path and allow long distance interactions
here is an example of how the enhancer region can be brought to close proximity with the promoter region
the protein binding the enhancer region can now interact with TF or mediators
Insulator
Euchromatin
Insulator
Heterochromatin
Role: Prevent spreading of heterochromatin to transcriptionally activ regions
chromatin domain are separated by DNA sequences called insulators
Trans acting factors are maintaining the structure of the insulator region in the proper configuration avoiding loss of cohesion
the euchromatin is organized in large loops that can undergo torsonial stress (changes in topology) that can be important for chromatin remodeling
Chicken B-globin gene cluster
Proteins such as CTCF (CCCTC-binding factor, a zinc-finger protein) can bind to the 5’ hyper=sensitive region HS4 preventing “chromatin invasion”
Concept of Boundary elements where a specific region prevents heterochromatin from invading the region that needs to be transcribed
Boundary elements are cis elements (sequences) that can be bound by specific DNA-binding proteins (example for beta globin: CTCF is a cCCTC-binding factor (zinc finger protein)
DNase insenstive = Low histone acetylation
DNase senstive = high histone acetylation
regulation of transcription in bacteria
Metabolism of lactose
Lac Operon: lactose is a precursor of glucose
regulation of transcription based on need for metabolism intermediates
Glucose requred will necessitate the lactose to be broken into glactose plus glucose
then enzyme required is the B-galactosidase
Lac Operon
- In the absence of lactose with a repressor = no lac nRNA
I, p, repressor, z, y, a
- Presence of lactose
I, p, o, z, y, z
z=galactosidase, y=permease, a=transacetylase
Transition for actively binding repressor vs inactiv binding
Positive Control of the lac Operon by glucose
POSITIVE CONTROL:
- The production of B-Galactosidase is controlled at the level of the Promoter-Operator region (operon).
- The lac z gene codes for ß-galactosidase
- Transduction of the signal for LOW GLUCOSE is transduced to an enzyme named ADENYLATE CYCLASE which role is to hydrolyze ATP to cAMP plus P~Pi (pyrophosphate).
- cAMP will form a complex with the protein CAP (not to be confused with the cap structure at the end of mRNA 7 methyl guanosine).
- The complex will bind DNA and the bacterial RNA polymerase at the Promoter-operator site.
CAP: Catabolic Activator Portein
P: promoter region
O: operator
Z: B-galactosidase
Negative control of the lac Operon
- NEGATIVE CONTROL: Repression.
- In presence of Lactose, no need for
- The expression of the structural genes is not only influenced by the presence or absence of the inducer, it is also controlled by a specific regulatory gene. The regulatory gene may be next to or far from the genes that are being regulated.
- The regulatory gene codes for a specific protein product called a REPRESSOR.
- The repressor acts by binding to a specific region of the DNA called the operator which is adjacent to the structural genes being regulated.
- The structural genes together with the operator region and the promoter is called an OPERON.
- However, the binding of the repressor to the operator is prevented by the inducer and the inducer can also remove repressor that has already bound to the operator.
- Thus, in the presence of the inducer the repressor is inactive and does not bind to the operator, resulting in transcription of the structural genes.
- In contrast, in the absence of inducer the repressor is active and binds to the operator, resulting in inhibition of transcription of the structural genes.
- This kind of control is referred to a NEGATIVE CONTROL since the function of the regulatory gene product (repressor) is to turn off transcription of the structural genes.
Identification of Eukaryotic Regulatory Sequences
- The DNA sequences required for proper activation and/or repression have been determined by deletion or addition of DNA within or adjacent to the promoter region(s).
- The regulatory sequence is cloned upstream of a reporter gene, the expression of which can be monitored by colorimetric assay (CAT or Chloramphenicol Acetyl Transferase assay), Northern blot of fluorescence measurements (GFP for example)
- The DNA was cloned into specific vectors (plasmid that can be expressed in Eukaryotic cells).
- Note that the plasmid is not integrated into the chromosome (transient expression will be lost when the plasmid is lost).
By changing the regulatory sequence one can determine the necessary region for proper regulation (ie the proteins that will bind to those sequences)
DNA Methylation
In females the X-chromosome exist in duplicate (XX vs XY)
If both copies of the X-chromosome were active, there would be an imbalance in expression of the genes coded by the X
One of the two X-chromosome is therefore inactivated
The inactivation mechanism involves DNA modification and protein binding to various regions of the X-chromosome
DNA methylation of Cytosine (in CpG dinucleotides) acts as a flag for gene inactivation
Note that specific protein can bind to methylated C (ex. MeCP2 or other MDBs)
Role is to maintain repression of the methylated DNMA regions
Overview processing eukaryotic mRNAs
modifications of 3’ ends of eukaryotic mRNAs
Consensus sequence for intron splicing in higer eukaryotes
Consensus sequences at donor and acceptor sites plus presence of the branching point (A).
Donor obeys the GT-AG rule: GT at the 5’end absolutely required) same for AG at the 3’end. The rest of the requirement for sequences is more loose.
Distance bewteen branching point and acceptor site is 20-40 ntdes.
Splicing reaction: generation of Lariat
Splicing requires the formation of a specific structure and sequential cleavage.
The 5’ site (acceptor is cleaved first and will be transiently ligated to the A at the branching point (forming a lariat structure). After that the 3’ site will be cleaved resulting in the removal of the intron. The Exons will be re-ligated.
Branch Poiint
The branching point displays a specific ligation motif. The nucleotide at the 5’ end will form a 5’-2’ bond between the G (5’) and the A(2’ at the branching point).
Nuclear splicing is a 2 reaction process in which a free OH end attacks a phosphodiester bond
The free OH attacks the 5’ phosphate of the G at the acceptor site. Then Exon 1 OH will attack the Phosphate at the Donor site of Exon 2
RNA splicing is Catalyzed by a spliceosome
After U1 sn RNA plus proteins forms U1 sn RNP binding at the 5’site, U2 sn RNA will bind to the branching point (through specific base-pairing at the Py tract). The complex U4/6/5 will
bind to the 5’ site and displace U1. SR splicing factors bind to specific sequence within exons, help recruit U1, U2AF and U2.
How is the lariat-like intermediate formed?
- U4/U6 complex will interact with U2, U5 is brought in contact with the 3’ site (AG sequence).
- The RNA is cleaved and the exons 1 and 2 are ligated.
- Note that Splicing Factors or SR proteins are present at the boundary of exons 1 and 2.
- U5 binds exon #1 at 5’ splice site
- U4/U6 binds U2
- 1st OH attack by U2/U6
- formation of lariat like intermediate
- U5 shifts, binds exon #2 at 3’ splice site
- U2/U6 catalyze 2’-5’ bond
- excision of intron
- ligation of exons
Group I and Group II introns are self-splicing
Self-splicing RNA (mitochondria, chloroplast, bacteria)
In bacteria and some organelles, the splicing mechanism exists in two different forms.
Group I: No lariat structure is formed, no A at the branching point. A guanosine substitute for the need of the A branching point.
Group II are similar to what was described in Eukaryotes.
Processing of rRNA
processing of tRNA
mRNA degradation
