Chapter 6. How Cells Read the Genome: From DNA to Protein. Test Your Knowledge. What are two major differences between transcription in prokaryotic and eukaryotic cells?
Video overview of transcription
“The Protein Players” - RNA polymerases, transcription factors, initiation factors, enhancers, repressors
Prokaryotic transcription video
Gene Regulatory Networks – control the number and type of transcripts made by a cell.
Simple core promoter
UAS = upstream activator sequence RE = regulatory elements INR = initiator sequence DPE = downstream promoter elements
General (or Basal) Transcription Factors:
TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH
Transcription Factors that Bind to Regulatory Elements
Recognizes and binds to TATA box; TBP + 10 TBP associated factors; position set
Binds and stabilizes the TFIID complex
Recruits RNA pol II + TFIIF to the location
Two subunits - RAP38 & RAP74. Rap74 has a helicase activity; RAP38 binds RNAPolII
Two subunits - recruits TFIIH to the complex thereby priming the initiation complex for promoter clearance and elongation
complex of 9 subunits. One w/ kinase activity; one w/ helicase activity; one is a cyclin (cdk7)
CTD of large subunit of Pol II
initiation complex, DNA melted at Inr
Sequential Binding Model for assembly of preinitiation complex
Polymerization of 1st few NTPs and phosphorylation of CTD leads to promoter clearance. TFIIB, TFIIE and TFIIH dissociate, PolII+IIF elongates, and TFIID + TFIIA stays at TATA.
Transcription initiation in the cell often requires the local recruitment of chromatin-modifying enzymes, including chromatin remodeling complexes and histone acetylases - greater accessibility to the DNA present in chromatin
Phosphorylation of the carboxy terminal domain (CTD) of one of the subunits of RNA PolII;
RNA polymerase II dissociates from the transcription factors and other protein complexes that were required for assemblyand elongation begins
Phosphorylation also promotes the accumulation of elongation factors – other proteins that arrest transcription long enough to recruiting RNA processing enzymes
Video of transcription and capping
CBC – cap binding complex proteins also associate and protect the cap;
Later they will direct transcript in its exit from the nucleus
R=A or G,Y=C or U
Introns are small-large;
Exons are about 150bp long
Exons might be easily identified, while introns probably couldn’t be.
As the RNA is being transcribed, SR proteins (rich in serine (S) and arginine (R)) sit down on the exons. Along with the U proteins, demarcates the start and end of the exon.
Capping proteins or polyA binding proteins act as markers at either end of the transcript.
Other hnRNPs (heterogeneous nuclear RNPs) bind along the introns, helping to distinguish these sequences from exons.
Changes in splicing patterns caused by random mutations have been an important pathway in the evolution of genes.
CstF = Cleavage Stimulation Factor
All of the A nucleotides are derived from ATP
Poly A binding proteins remain until mRNA undergoes translation
Guided diffusion along the FG-repeats displayed by nucleoporins
Proteins bound to mature mRNA molecules and that signal completed splicing have nuclear export signals as a part of their sequence
hNRPs “straighten out” the mature mRNA so that nuclear export signals can be “read”
This is the only tRNA that can bind to the small ribosomal subunit by itself
New tRNA carrying amino acids are accompanied by elongation factor called EF-Tu
The tRNA-ETu occupies a hybrid binding site (not quite in A)
Correct codon-anticodon pairing triggers ETu to split GTP and fall off, and tRNA moves into the A position
The delay caused by the association/dissociation of ETu helps increase accuracy of translation
Elongation factor G (EF-G) then binds near the A site, forcing the tRNAs containing the new amino acid and the growing chain into the next (P and E) sites on the ribosome
EF-G splits GTP, changes conformation and falls off, thus increasing the speed of translation.
GTP exchange factors continually recharge the GTP on both of the elongation factors.
No tRNA binds to this set of codons
One of these codons at the A site attracts a release factor
Ribosome adds a water to the last peptide, creating the carboxyl end
Hsp 60 works like a quality control chamber after a protein is completely folded
Cap hydrolyses ATP; regulates exit
Proteasomes are a major mechanism by which cells regulate the concentration of particular proteins and degrade misfolded proteins.
Proteins are marked for destruction by the addition of a small molecule called ubiquitin on exposed lysine residues
Cells can also regulate protein degradation by activating new ubiquitin ligases via different mechanisms.
Proteins generally “hide” degradation signals so that they are not active. However, there are several mechanisms for exposing the degradation signals.
You have isolated an antibiotic named edeine, from a bacterial culture. Edeine inhibits protein synthesis but has not effect on either DNA synthesis or RNA synthesis. When added to a reticulocyte lysate, edeine stops protein synthesis after a short lag, as shown below. By contrast, cycloheximide stops protein synthesis immediately. Analysis of the edeine-inhibited lysate by density-gradient centrifugation showed that no polyribosomes remained by the time protein synthesis had stopped. Instead, all the globin mRNA accumulated in an abnormal 40S peak, which contained equimolar amounts of the small ribosomal subunit and initiator tRNA.
Radioactivity in hemoglobin
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