Bmsc 220 6b Transcription and RNA processing
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to transcribe, RNApolymerase II requires that a gene contains:
- -exons (coding);
- -introns (noncoding);
- -a stop sequence
- -core promoter region (Inr and TATA or TATA-like box;)
proteins that help RNA polymerase II transcribe genes
general transcription factors
- -core promoter and INR
- -essential for RNA pol II binding
gene-specific transcription factors
- -not part of the general machinery
- -bind to promoter and enhancer elements
- -control the rate of transcription
- -upstream promoters
general transcription factors that bind to TATA sequence before and after RNA pol II does
- before: TFIID TFIIF
- after: TFIIE and TFIIH
What must happen before RNA pol II can bind to the TATA sequence?
what happens before transcription startes
-general transcription factors called TATA-binding proteins (TBPs TFIID andT FIIF) must bind to the TATA box.
-After RNA pol binds, two more TBPs bind (TFIIE and TFIIH), and transcription can start
Gene-specific promoter and enhancers
- -proteins like transcription factors
- -bind to promoter and enhancer sequences on the DNA to alter the rate of transcription (by affecting the efficiency of the pre-initiation complex).
-either downstream or upstream of transcription start site
-bind transcription factors which ↑ or ↓ the amount of the transcript (mRNA)that is made
– modulate the rate of transcriptional regulation by regulating the pre-initiation complex
•Mutations in regulatory DNA sequences or transcription factors leads to:
- human disease
- sequences: beta thalassema
factors: Rett syndrome
- -have a modular structure
- -Can function as transcriptional activators and transcriptional repressors
-contain separate DNA-binding and activation/repression domains
-contain other domains that regulate their activity or allow them to interact with other transcription factors
Mediator protein complex
- - large protein complex
- -plays a key role in linking the general transcription factors to the gene-specific transcription factors
- – binds to the pre-initiation complex
- -DNA folds on itself
- -allows transcription factors bound to a distant enhancer to interact with proteins in the RNA polymerase/ Mediator complex at the core promoter
Release of RNA polymerase from the basal (pre initiation) complex to initiate transcription requires:
the phosphorylation and helicase activities of TFIIH:
-Phosphorylation of C-terminal domain (CTD) on RNA pol II
-Helicase must break hydrogen bonds in dsDNA for RNA pol II to start working
2 general mechanisms of action
- 1. Interact with mediators and general transcription factors
- 2. Interact with co-activators, which modify chromatin structure.
-Blocking binding of transcriptional activator to DNA sequence element
-Repressing mediator activity
-Interaction with co-repressors, which serve to modify chromatin structure into a non-permissive state
•Actively transcribed chromatin exists in a relatively ___ state
- a) Histone tail acetylation and deacetylation
- b) Other histone modifications
Histone acetylase (HAT)
- histone tail acetylation:
- -addition of acetyl groups to specific lysine residues in histone tails.
- -transcriptionally permissive chromatin
Histone deacetylase (HDAC)
- histone tail deacetylation (HDAC):
- non-permissive state
- -must bind to activator first
- -must bind to either a repressor first
Chromatin remodeling factors
-increase availability of a gene to be transcribed by displacing the nucleosome
- •processing of the pre-mRNA to the mature RNA
- -occurs in the nucleus
- -allows for extra level of regulatory control
Processing of Eukaryotic messenger RNAs
- 5' capping
- 3' polyadenylation
the primary transcript that is processed to form messenger RNA in eukaryotic cells.
-A 7-methylguanosine cap is first added during the modification of the 5' end of a transcript, often before transcription has ceased.
-increases stability (“half-life”) of mRNA (makes the 5’ end look like a 3’ end so them RNA will not be degraded by 5’ exonucleases)
-addition of a Poly-A tail to 3' end of eukaryotic mRNAs by the enzyme poly-A polymerase.
-protectsthe mRNA molecule from enzymatic degradation in the cytoplasm, aids intranscription termination, helps the export of mRNA from the nucleus
a tract of about 200 adenine nucleotides
What directs where Poly A will occur?
A DNA sequence along with a cleavage by endo nuclease
Small nuclear RNAs (snRNAs)
- -nuclear RNAs that range in size from 50 to 200 bases and form the RNA component of the spliceosome.
- -assist in targeting snRNPS to splice junctions through hybridization to RNA transcript
- -are catalytic and carry out steps in the splicing reaction
- U1, U2,U4, U5, and U6 snRNAs
•Small nuclear ribonucleoprotein particles (snRNPs)
- complexes of snRNAs with proteins that play central roles in the splicing process.
- U1, U2, U3, U5, and U4/U6 snRNPs
- -are large complexes composed of proteins and RNAs (snRNAs)
- -carry out the splicing reactions
SR slicing factors
- -bind to exon sequences to direct where a splicesome will form
- -will differ in two different cell types for which a specific mRNA is alternatively spliced
- -assist in determining the final “choice” of exons that will appear in any one cell type for any one alternatively spliced mRNA
- -generation of different mRNAs by varying the pattern of pre-mRNA splicing
- -allows many different combintions
- -allowed by presence of introns between exons in a gene
- •In the sex determination mechanisms of Drosophila, alternative splicing of the same pre-mRNA determines whether a fly is male or female.
-expressed as a cell surface adhesion molecule(marker) important in allowing neurons to find their target cells
-contains four sets of alternative exons, with a single exon from each set being incorporated into the spliced mRNA.
5.8S, 18S, and 28S rRNA: (RNA pol I)
- -makes up the majority of cellular RNA (over 80% of total cellular RNA)
- -are the products of one unique gene (45S rRNA gene) that is present in multiple repeated copies within the genome
the primary transcript that is cleaved to form individual ribosomal RNAs—the 28S, 18S, and 5.8S rRNAs of higher eukaryotic cells.
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