Lecture 28 Gene Production of Proteins Flashcards
How do cells achieve their specialized functions?
- Based upon the expression of specific genes and any subsequent post-translation modifications of the resulting proteins
Translation
- RNA to protein
Transcription
- DNA to RNA
Eukaryotic Protein production
- Transcription in nucleus
- 5’ capping RNA splicing
- 3’ polyadenylation
- Exported to cytoplasm
- Translated into protein
Prokaryote protein production
- Transcription into mRNA
2. Simultaneous translation into protein
Introns
- are transcribed but not translated
What is different in deoxyribose from ribose?
The hydroxyl group on 2’
How many strands of DNA is transcribed into RNA?
one
What direction is RNA synthesized?
5’ to 3’
What direction is the DNA template strand oriented in?
3’ to 5’
Promoters
- sequences in the DNA that promote or direct transcription of a gene
What are most protein coding genes transcribed by?
- RNA polymerase II
Transcription
- initiates at a specific point in the DNA and requires unwinding of the DNA to create the proper single-stranded template
Gene expression regulatory proteins
- recognize specific DNA sequences and upon binding regulate if, when, and to what extent a gene is transcribed.
Can you have multiple RNA polymerase molecules transcribing a gene?
- Yes
mRNA
- messenger RNA
- Translated into proteins
tRNA
- transfer RNA
- Transfer amino acids to the growing peptide chain
rRNA
- ribosomal RNA
- Encodes ribosomal proteins
microRNA
- Block translation of specific mRNAs and thereby regulate gene expression (at post translational level)
- new
- Last 10 years
siRNA
- small interfering RNAs
- turn off gene expression by directing the selective degradation of mRNAs.
snoRNA
- small nucleolar RNA
- Process and chemically modify rRNAs
what is the most abundant RNA?
- ribosomal RNA (80%)
Where is the TATA located?
- 25 base pairs upstream or downstream from start cite of transcription
What does TATA box do?
- Phases binding proteins that bring in RNA polymerase so that it starts at the right spot
What binds to TATA box first?
TFIID
- has part called TBP that binds it at the right spot
TFIIB
- binds after TFIID
What is required for TFIID and TFIIB to bind?
- must have regulatory transcription factors present for the process to initiate
TFIIH
- important transcription factor that has heliocase activity
What does phosphorylation of C terminal domain of the RNA polymerase do?
- open the polymerase to an active conformation for transcription
- some transcription factors will disassemble
TFIID
- recognizes the TATA box
- has subunits (TBP)
RNA pol I
- transcription of rRNA genes,
RNA pol III
- transcribes tRNA genes, 5S rRNA
RNA processing
- Capping
- Splicing
- Editing
- Polyadenylation
- Transport
When does splicing take place?
- as transcription is proceeding
Where is the cap located?
- 5’ end
Where is the Poly A tail located?
- 3’ end
Spliceosome
- performs splicing
- made up of snRNAs in complex with 7 protein subunits to form SNP
snRNAs involved in splicing
- U1
- U2
- U4
- U5
- U6
What do snRNAs do in splicing?
- provide for proper base pairing with the mRNA
- These RNA-RNA arrangements are dynamic and shift throughout the splicing process
When does 5’ capping occur?
- as soon as the hnRNA emerges from the RNA polymerase
- could be before splicing has been done
When is the poly A tail added to mRNA and what adds it?
- as soon as it emerges from the RNA polymerase
-
What is microRNA?
- small non-coding RNA
- 21-25 nt in length
- bind to 3’ UTR region of target to form and RNA-inducing silencing complex
What does microRNA do?
- Suppresses protein synthesis and or induce mRNA degradation
- Each miRNA can target up to 100 different mRNAs
codon
- 3 base sequence that codes for one amino acid
Important features of the genetic code
- comma-less (read from beginning to end so reading frame is critical)
- degenerate (more than one codon can make the same amino acid)
- The third base in the triplet codon is less specific than the first tow
- 3 of 64 possible codons do not code for amino acids but signal termination
AUG
- codes for methyleline and is where translation starts
What are the stop codons?
- UAA
- UAG
- UGA
Where are ribosomes assembles?
- Nucleus (nucleolus)
- Nucleolus is not a membrane bound structure
SnoRNAs
- serve as guide RNAs to direct specific modifications of the RNAs
- These modifications include methylations, and isomerizations
Robert syndrome
- mild retardation
- retardation, craniofacial abnormalities
- short arms/legs
- decrease in rRNA leads to decreased protein synthesis
Roberts syndrome
- decreased protein synthesis of rRNA genes
- Homozygous mutation of ESCO2 which encodes an acetyltransferase important for the foramtion of the cohesion complex that binds to chromosomes and creates cohesion between sister chromatids.
Free ribosomes
- move anywhere in the cytosol but are not found in the nucleus and other organelles
Membrane bound
- if the protein being made contains an ER targeting sequence then the ribosome is associated with the ER in the rough ER
- These types of proteins are transported to their destination through a secretory pathway and are usually associated with the plasma membrane or secreted out of the cell
Translation
- mRNA is read by the ribosomal machine as a triplet of sequential nucleotides (called a codon)
- Translation starts at the 5’ end of the mRNA
- tRNAs are “charged” by the addition of a specific amino acid that corresponds to that codon. this aminoacyl-tRNA is created by the action of enzymes called aminoacyl-tRNA synthase
- Protein translation uses base pairing between the mRNA codon and a triplet complementary sequence in the tRNA called the anticodon
Four steps of translation
- Activation
- Initiation
- Elongation
- Termination
Activation
- formation of aminoacyl- tRNAs
`Initiation
- binding of small ribosomes to 5’ end of mRNA and subsequent binding of initiator Met-tRNA
Elongation
- synthesis of the peptide chain
Termination
- synthesis stops and peptide (protein) is released from the ribosome
What is the initiator of translation?
- Met-tRNA
When does translation stop?
- when two stop codons are encountered in the mRNA
Many antibiotics target transcription or translation
- rifamycin
- tetracycline
- streptomycin
- chloramphenicol
- erythromycin
Rifamycin
- prevents RNA synthesis
Tetracycline
- Blocks binding of the aminoacyl-tRNA to the A-site (also binds to newly forming mineralizing surfaces such as bone and teeth)
- causes staining
- don’t use in children bc of staining
Streptomycin
- prevents the switch from translation initiation to elongation and also can cause protein miscoding
Chloramphenicol
- blocks the peptidyl transferase reaction so elongation is prevented
Erythromycin
- blocks the ribosomal exit channel in the ribosome so elongation is inhibition