Lecture 5A - Transcription Flashcards
What does transcription of DNA yield?
A single stranded RNA that is complimentary to one strand of DNA (template strand)
What are the general features of transcription? (what, where, similarities/difference b/t RNA and DNA synthesis)
Transcription: transfer of genetic information from DNA to RNA
- Occurs in the nucleus
- Ribonucleoside triphosphates monomers
- Only one strand of DNA is template for RNA
- No primer required => de novo synthesis
- RNA is unstable, short-lived compared to DNA
Why is relative instability of RNA a good thing?
Serves as a means of regulation and gene expression. It can be protected from degradation by RNA binding proteins => regulate RNA stability
Features of RNA produced from transcription? 5 types of RNA?
- Single stranded
- Complementary to DNA template strand, identical to DNA nontemplate strand (thymidines replaced w/ uracil)
5 types:
- Messenger RNA (mRNA)
- Transfer RNA (tRNA)
- Small nuclear RNA (snRNA)
- Ribosomal RNA (rRNA)
- Micro RNA (miRNA)
What are RNA polymerases? (direction, nuc attack, helicase or no, primer or no)
- Enzymes that catalyze the synthesis of RNA complement to DNA
- 5’ to 3’ direction
- Nuc attack by 3’ OH on nucleotidyl phosphorus phosphorus atom w/ elimination of pyrophosphate
- Localized unwinding and rewinding DNA w/o need for helicase
- No primer required to initiate polymerization
RNA transcription occurs w/in a ______
locally unwound segment of DNA
What is a transcriptional unit?
A segment of DNA transcribed to produce one RNA molecule; one or several genes on a single RNA molecule
What is a gene?
A unit of genetic information that controls the synthesis of one protein or structural RNA molecule (RNA that is not mRNA)
What is meant by downstream/upstream?
Downstream: towards 3’ region of gene
Upstream: towards 5’ region of gene
What is sense/antisense RNA strands? Sense/antisense DNA strands? Relation to template/nontemplate DNA strands?
Sense RNA strands encode nucleotides that specify AA of gene products (ex: mRNA)
Antisense RNA strands are complementary to sense strands (ex: miRNA, long noncoding RNA => regulate expression of sense RNA by binding to sense strand as complementary sequence)
Sense DNA strand (nontemplate) is complimentary to antisense DNA strand (template), and identical to sense RNA strands
Overview of eukaryotic transcription: 6 features
MORE COMPLEX THAN PROKARYOTIC
- mRNA synthesized in nucleus
- Proteins synthesized in cytoplasm
- Prokaryotic transcription often results in multigenic mRNAs (gene clusters/operons, genes w/ shared function for a process are transcribed in the same piece of RNA)
- Eukaryotic transcription often results in monogenic mRNAs
- 3 RNA polymerases
- 3 modifications of mRNA that help w/ translation and stability
- 7-methyl guanosine cap on 5’ end
- Poly-A tail on 3’ end
- Splicing out introns
What are peri and posttranscriptional processing? Where do they occur?
Peritranscriptional processing: modification occurs while transcription is occuring
Posttranscriptional processing: modification occurs on mature mRNA after RNA has been synthesized/transcription complete
All occur in nucleus
3 types of peri-/posttranscriptional processing
- 5’ end 8 methyl-guanosine cap (peri)
- 3’ end polyadenylation (Poly-A tail) (post)
- Introns spliced out: non-coding intervening sequences
What is hnRNA? Where does it go? How are transcripts protected from RNases? What is the relative half life of eukaryotic vs prokaryotic RNA?
hnRNA: heterogeneous nuclear RNA i.e. primary transcripts in the nucleus that can have diverse function ((snRNA, rRNA, tRNA, mRNA, etc.)
- Exported to cytoplasm for translation
- Protected from RNases by RNA binding protein coating, which regulates stability of RNA
- Eukaryotic RNA has a longer half life than prokaryotic RNA
How many RNA polymerases in eukaryotes? What do they do?
Three; RNA polymerase I, II, and III
RNA polymerase I transcribes 5.8S, 18S, and 28S rRNA genes (ribosomes)
RNA polymerase II transcribes all protein-coding genes, snoRNA genes, miRNA genes, siRNA genes, and most snRNA genes
RNA polymerase III transcribes tRNA genes, 5sRNA genes, some snRNA genes, and genes for other small RNAs
How many RNA polymerases in prokaryotes?
One
What cofactors RNA polymerases require to initiate transcription? Eukaryote vs prokaryote
Eukaryote: many additional proteins called general transcription factors
Prokaryote: single additional protein called sigma factor
What do eukaryotes have to deal w/ in transcription initiation that prokaryotes don’t have to?
Packing of DNA into nucleosomes and higher order forms of chromatin structure
What are the three stages of transcription?
- Initiation
- Elongation
- Termination
Eukaryotic Transcriptional Initiation
- Requires transcription factors
- Binding of polymerase complex to promoter
- Unwinding of DNA
What is a promoter sequence? What is the structure of promoter recognized by RNA pol II?
DNA sequences where TF bind to
TATA box
What are general transcription factors?
- Proteins required for initiation of transcription by RNA Pol II
- Denoted TFII-X (TFII = TF for Pol II)
- Bind promoter sequences inn a specific order
What initiates transcriptional elongation?
The phosphorylation of Pol II “tail” triggers escape from initiation complex
What is the direction of transcription determined by? Can transcription occur off of either strand of DNA or just one?
Direction: determined by promoter at beginning of each gene
Can occur off of either strand
What does DNA bending by transcription factors do?
Serves as recruitment site for other proteins that take part in transcription
What is the activator protein? How does it get brought to promoter and initiation complex?
A protein that activates/enhances transcription; bound to enhancer site typically far from the promoter
DNA can be coiled (chromatin packaging), bringing activator proteins close to initiation complex
What does the initiation complex consist of?
- Mediator (binds activator)
- Chromatin remodeling complex
- Histone modifying enzyme (HATs and HDACs)
- RNA processing enzymes
- RNA polymerase
Describe elongation by RNA pol II (euk vs prok, where does it occur, primer or no)
- Same in proks and euks
- Occurs w/in transcription bubble
- RNA pol unwinds and rewinds DNA
- No primer required
- Short RNA/DNA heteroduplex
What recruits RNA processing enzymes? How?
- The tail of RNA pol II
- Phosphorylation state of tail determines which factors are recruited
What happens to 5’ end during elongation? When? Why?
- Peritranscriptional modification => pre-mRNA gets 7-MG cap (unusual 5’-5’ triphosphate bond)
- Added when chain is ~30 nucleotides in length
- Function: recognized by posttranslation machinery and protects 5’ end from degradation
Describe transcriptional termination (Polyadenylation and cleavage, why polyadenylate?)
- Poly (A) polymerase adds ~200 A’s to 3’ end
- Coupled w/ cleavage by endonuclease, as polyadenylation occurs right after cleavage
- Polyadenylation increases stability
What are mRNA?
Messenger RNAs, code for proteins
What are rRNAs?
Ribosomal RNAs, form basic structure of ribosome and catalyze protein synthesis
What are tRNAs?
Transfer RNAs, central to protein synthesis as adaptors b/t mRNA and AA
What are snRNAs?
Small nuclear RNAs, function in a variety of nuclear processes, including the splicing of pre-mRNA
What are snoRNAs?
Small nucleolar RNAs, used to process and chemically modify rRNAs
What are scaRNAs?
Small cajal RNAs, used to modify snoRNAs and snRNAs
What are miRNAs?
Micro RNAs, regulate gene expression typically by blocking translation of selective mRNAs
What are siRNAs?
Small interfering RNAs, turn off gene expression by directing degradation of selective mRNAs and the establishment of compact chromatin structures
What are noncoding RNAs?
Function in diverse cell processes, including telomere synthesis, X-chromosome inactivation, and the transport of proteins into the ER
Eukaryotic transcription occurs in _______ and translation occurs in ________
Nucleus; cytoplasm
Prokaryotic transcription/translation not separated by cellular compartments, often coupled
True
False
True
Prokaryotic mRNA is _____, allows for coordinated expression for genes w/ same function
polycistronic
Eukaryotic genes have both expressed and non-expressed sequences - exons and introns|describe
Exons: sequences that remain in mature mRNA
Introns:
- Intervening sequences that are spliced out,
- Found in most eukaryotic genes
- Variable in size
- Some sequences w/in introns regulate gene expression
- Mutations occur more rapidly
Exons have both coding and noncoding regions, why?
Noncoding region is where ribosome binds, esp 5’ and 3’ untranslated regions
What is exon shuffling? What is alternative splicing?
Exon shuffling: the rearrangement of exons for the evolution of “new” exon combos
Alternative splicing of pre-mRNA: one gene but different transcripts, can modify coding sequence by removing exons
Splicing exons together requires highly precise removal of each intron - why?
One base addition/deletion will make huge difference => nonfunctional protein, different ORF, etc.
What are spliceosomes? Two steps to splicing?
Complex RNA/protein structure that carries out RNA splicing
Composition:
- >40 proteins
- numerous snRNA (small nuclear RNA)
- proteins + snRNA complexed as snRNP
- snRNP form spliceosome core
Two steps:
1. Cleavage
2. Ligation
Outline of pre-mRNA splicing process
- Cleave 5’ end
- Form lariat
- Cleave 3’ end
- Release lariat from pre-mRNA
- Splicing of adjacent exons (ligation)
- Lariat degraded, snRNP recycled
(steps 3-5 are simultaneous)
(refer to slide 51 lec 5A)
Splicing abnormalities result in ____
Mutations
What is the nucleolus the site of?
Nucleolus is the site of rRNA and ribosome synthesis
