General Genetics Exam 4

Occurs in early development •Same set of genes being used differently accounts for the phenotypic differences - along with mutations - that occur outside of the genome •gene expression at certain times •controlling the expression of genes in TIME (eg. developmental stage) and SPACE (eg. tissues, cells, etc.)

DNA •Thymine •Double-stranded RNA •Uracil instead of Thymine •2’ OH group instead of H (in the sugar of the sugar-phosphate backbone) for easier degradation •single-stranded (but associates with itself to form double-stranded regions) •Only has primary and secondary structure • exists only as a secondary (looping) structure w/i cell • tRNA has a distinct secondary structure (allows binding to codon and mRNA)•Ribose sugar

Forms very complex higher-order structure (not restricted to double helix) •looping structure •complementary sequences associate to form a double-strand in some regions
tRNA has a distinct secondary structure (allows binding to codon and mRNA)
DNA is more restricted due to the double-stranded helix, so it has fewer functional roles in the cell

Prokaryotes and Eukaryotes: mRNA: primary transcript formed during transcription of a gene itself; protein coding sequence rRNA: complex of a ribosome tRNA: bring amino acids to ribosome during the elongation of the polypeptide chain during translation Eukaryotes only: snRNA (small nuclear RNA): pre-mRNA processing (splicing) - there are disruptions in coding region that need to be spliced out (by snRNAs); snRNP miRNA (microRNA): seeks out and binds to complementary sequence mRNA, turns off transcription of that mRNA, a way for regulation of translation siRNA (small interfering RNA): trigger degradation of target mRNAs, used by many viruses

1. 1. Transcribed strand of DNA
2. 
   
3. 2. RNA synthesis runs in 5’ – 3’ direction (nucleotides added to 3’ end)

•synthesis is complementary and antiparallel to template

3. 3. Either DNA strand can be used as a template (no real preference)

•however, transcription always occurs from 3’ – 5’ end of DNA

4. 4. The direction/the overlapping of genes in DNA determine which strand will be used as the template to transcribe the gene from
5. 
   
6. 5. Yes, units can overlap because RNA polymerases can synthesize on either DNA strand

Promoter: a DNA sequence recognized and bound by the transcriptional apparatus •core sequences recognized by the RNA Polymerase holoenzyme •”upstream” of the RNA coding region; is immediately 3’ to RNA start site
Indicates: 1. which strand will be used as a template 2. which direction transcription will occur 3. transcription start site RNA-coding region: encodes for RNA molecule itself
Terminator: termination sequence that ends transcription; at 5’ end of template

1. Transcription in prokaryotes 2 sequences w/i promoter: recognized by RNA polymerase (explains why RNA poly chooses to use that strand as a template over the other strand) •TATAAT box (Pribnow box) •TTGACA sequence •2 sequences have to be close on the template strand in specific order for RNA poly to recognize and bind to the promoter sequence; defines directionality of transcription 2. RNA polymerase holoenzyme recognizes the elements

Sigma factor: recognizes the sequences in the promoter and is responsible for the binding of RNA polymerase to the strand •initiates transcription • specificity of binding to DNA requires sigma •Without sigma, RNA polymerase will initiate transcription at a random point along the DNA •stays associated w/ RNA poly until RNA poly completely associates w/ strand once it starts elongation •only required for recognition and initiation •5 subunits (with sigma) form holoenzyme

Prokaryotes
Initiation:

1. -sigma factor on RNA poly binds to promoter sequence
2. -Holoenzyme associates with sigma factor (transcription bubble forms)
3. -Formation of initial rNTP bonds
4. -Sigma factor falls off RNA polymerase once it clears the promoter (this is where enhancers can come into play and help RNA poly stay associated with template strand

Elongation:

1. -Sigma comes off and RNA poly changes shape
2. -DNA poly works faster than RNA poly b/c of its higher processivity to template strand
3. -RNA polymerase has limited proofreading ability
4. -RNA Polymerase progressively unwinds DNA at leading edge of bubble.

Termination:

1. -Termination sequence = stops transcription
2. -Rho-dependent and independent

•independent: contain inverted repeats; Poly-A tail following second repeat •dependent: Rho binds to unstructured region upstream of terminator ~goes towards 3’ end, catches up with RNA polymerase, using helicase activity Rho unwinds DNA:RNA hybrid to release transcribed RNA

Initiation: 1. Basal apparatus (holoenzyme): •RNA Pol II, general transcription factors, mediator •Function of sigma replaced by general transcription factors -Binding of TFIID bends DNA partially unwinding it -binding of holoenzyme forms transcription bubble (11-15 bp) -TF2D includes TATA binding protein so it binds to the TATA box (acts like sigma factor) •required for transcription for the holoenzyme to recognize and bind to the template •forms pre-initiation complex that undergoes basal level of transcription (low slow level) •impoves efficiency during process of initiation
2. Enhancer elements: bound by protein transcription factor and causes DNA to loop back on itself and associate with the enzyme to transcribe the template at a more efficient rate •RNA Poly II to clear the promoter in order to enter elongation and increase processivity Elongation:

1. TF2H is a protein complex that acts as a helicase (forms transcription bubble)
2. Extension always occurs at 3’ hydroxyl group end
3. RNA Poly II then reaches a termination sequence
4. DNA/RNA duplex bent at right angle

Termination: -RNA Pol I uses termination factor like RhoAt end of transcriptinal unit, it transcribes past the end of the mRNA•end of transcription sequence is recognized by an endonuclease and cleaved at consensus sequence•exonuclease (Rat1) chews up at 5’ end to 3’ end at higher rate than RNA poly II can transcribe•once it reaches/catches up to the RNA poly II, it terminates transcription•exonuclease activity is specific to RNA poly II to terminate transcription (rho is specific to prokaryotes)
-RNA Pol III uses mechanism like Rho-independent termination

Refers to termination sequence in prokaryotes
Rho-dependent:-Rho binds to unstructured region upstream of terminator -Rho goes towards 3’ end, catches up with RNA polymerase, using helicase activity Rho unwinds DNA:RNA hybrid to release transcribed RNA
Rho-independent:-contains inverted repeats; Poly-A tail following second repeat-reverse complementary sequences bind to form a stem loop that is high energy and hard to break-sequence is recognized by RNA polymerase where transcription will stop (doesn't recognize template, but structures and sequences that cause termination)

RNA Polymerase I: Larger rRNAs
RNA Polymerase II: pre-mRNAs, some snRNAs and miRNAs
RNA Polymerase III: tRNAs, small rRNAs and miRNAs

•The core promoter (TATA box) is -25 from transcription start site; where the basal transcription apparatus binds
•the regulatory promoter is immediately upstream from the core promoter -transcriptional activator proteins bind to the sequences and make contact with the basal apparatus and enhancers ~affects the rate at which transcription is initiated

Yes, the sigma factor is replaced by general transcription factors

RNA Polymerase II uses a mechanism similar to Rho-independent termination •When RNA is transcribed, as mRNA is exposed, it exposes particular sequences to endonuclease RNAse that cuts that DNA sequence •Rat 1 chews up RNA faster than poly II can make it; when Rat 1 reaches poly II, it terminates transcription
RNA Polymerase I uses a termination factor like Rho-dependent termination