RNA and Protein Synthesis Flashcards
What is a gene?
segment of DNA that contains all the information necessary for the synthesis of a functional product (protein or RNA)
What two things compose a gene?
- untranscribed promoter and enhancer regions necessary to regulate transcription
- transcribed region that contains exons, introns, and untranslated regions (UTR)
What are exons?
coding regions which are translated into proteins
What are introns?
non coding, intervening sequences, which are removed post transcriptionally by splicing
What are UTRs?
untranslated regions at the 5’ and 3’ ends which regulate translation and mRNA stability
Basic steps from transcription to translation?
- Exons and introns are transcribed
- 5’ cap and 3’ Poly A tail are added
- Introns are spliced out
- Transmembrane transport
- Translation
Does RNA Polymerase need a primer to begin transcription?
No it does not need a primer to provide the first 3’ OH, but by itself it binds DNA weakly and nonspecifically
What helps RNA polymerase to begin to transcribe in the proper location and the correct rate?
- Basal/General transcription factors
2. Transcriptional activators (factors, TSFs)
What helps RNA polymerase know where to start?
Basal/General TFs help to position it properly at the start site
What helps RNA pol be more efficient in transcription?
Transcriptional activators make other TFs more efficient
Where do Basal/General factors bind?
they bind as a multi protein complex to the TATA box
What helps separate the strand to make the transcription bubble?
General/ Basal factors
-they regulate low, basal level transcription
T/F
Basal/General factors are the same for every gene.
True
Are basal factors regulated by the environment?
No
What is the TATA binding protein (TBP) part of?
TFIID
Where does TBP bind to?
directly to the TATA box
What is the role of TBP?
the completion of initiator complex causes a conformational change , it kinks DNA to help separate the strands
What is the last of the basal TFs to bind the initiation complex?
TFIIH
What is TFIIH and what is its role?
- multisubunit protein
- helicase activity uses ATP to unwind the helix and separate strands of DNA
- signals to being transcription
- important in transcription coupled repair of DNA
What specifically signals the start of transcription in TFIIH?
kinase activity phosphorylates the C terminal domain of RNA pol II
How do transcriptional activators regulate gene expression?
by influencing the rate of transcriptional initiation
Where do transcriptional activators bind?
a certain nucleotide sequence in the DNA called a consensus site or enhancer element (in regulatory regions) via its DNA binding domain
-each gene is bound by a different set of activators, depending which enhancer binding sites are present
How do transcriptional activators domains affect the rate of transcriptional initiation?
facilitating the formation of the basal initiation complex
How is the initiation complex formed in transcriptional activators?
- recruiting HATs and chromatin remodeling complexes
2. interacting with general transcription factors directly or indirectly through proteins called mediators
What can pioneer factors do?
they can bind to condensed chromatin and help loosen it so other transcription factors can bind
T/F
Transcriptional activators are the same for every gene.
False
Are transcriptional activators regulated by the environment?
yes they abundance or activity are regulated by in response to environmental signals
How are pyrimidines indicated in consensus binding sequences? purines? What is N?
Pyr- Y
Pur- R
N- no particular base in more common
What happens if the DNA sequence conforms well to the consensus sequence?
- the binding affinity is higher
- genes with better binding sites will bind that transcription factor more readily than others if amount are limiting
What does the net effect of transcription factors determine?
rate of transcription determined by activators, repressors, and all other transcription factors
What are 3 things that change heterochromatin to euchromatin?
- coactivator complex
- Loss of H1
- Histone acetylation
What are 3 things that change euchromatin to heterochromatin?
- Corepressor complex
- deacetylation
- methylation
What is the role of repressors in transcription?
- inhibit the initiation of transcription
- can use more than one mechanism, depending on promoter context and circumstances
How do repressors interfere with the activity of transcriptional activators?
- compete for same binding site
- bind to and obstruct/mask the activation domain of an activator
- recruit histone deacetylases (HDACs) or other proteins that cause heterochromatin formation
What is the next step after initiation of transcription? What is the first thing that happens in this step?
- elongation
- the coding strand is held out of the way while the template strand is copied
-first nucleotide remains a triphosphate and is quickly protected by the addition of the 5’ cap (7 methylguanosine)
In transcription, what strand is the RNA sequence complimentary to?
template strand of DNA
-it is the same as the coding strand but with U instead of T
What are the major RNAs in protein synthesis?
mRNA- heterogeneous, as many kinds as cell makes protein
tRNA- 70-90 Nuc, 31 kinds
rRNA- 4 kinds, 3 from same precursor RNA, combine with over 80 proteins
How many major RNA polymerases are there?
3
RNA Pol I
RNA Pol II
RNA Pol III
What makes mRNA post transcriptional processing more efficient? what else might it do? What else might travel with RNA pol?
RNA pol II carries some of the pre-mRNA processing proteins on its phosphorylated C terminal domain, which are transferred to newly transcribed mRNA at the right time
- may help to prevent RNA from annealing to the DNA
- Some DNA repair enzymes might travel
What is the structure of the 5’ cap? what type of linkage does it make? What also gets methylated?
- 7 methylguanylate with a 5’ to 5’ linkage
- the first 1 or 2 ribonucleotides of mRNA are methylated
What adds the 5’ cap?
capping enzymes that are carried on the phosphorylated C terminal domain of RNA pol II
What are 5 functions of the 5’ cap?
- marks 5’ end of mRNA
- distinguishes mRNA from other kinds of RNA
- mRNA stability
- transport of mature mRNA from nucleus to cytoplasm
- necessary for efficient translational initiation
What are introns? When are they spliced? What is transported to the cytoplasm?
- intron-intervening sequences that do not encode protein
- they are removed in the nucleus
- exons, or the translated regions are spliced together before the mature mRNA is transported to the cytoplasm
What is the role of snRNAs? What are snRNPs?
snRNA- non coding RNA, small nuclear RNA, recognize the splice sites and do the splicing
snRNP- each snRNA is in a complex with several proteins to form small ribonucleoprotein particles (snRNPs)
Alternative splicing?
some mRNAs can be spliced in different ways to yield different proteins, almost 60% can be alternatively spliced
Explain why splicing is a difficult process? Why is the complexity of it important?
- splicing machinery is complex, involving 5 different RNAs, and several hundred proteins and uses a lot of energy in form of ATP
- complexity allows the mechanism to be accurate, but still flexible to deal with a wide variety of introns
In mRNA splicing, what defines the eons?
consensus sequences
What nucleotide almost always occurs between an exon and an intron?
GG
How many genetic diseases are due to mutations in splice sites?
10-15%
Where is mRNA cleaved at the end of transcription?
downstream of the poly Adenylation signal (AAUAAA)
What is the role of Poly A Polymerase? Then what binds?
- adds 200 A’s to the 3’ end of mRNA
- poly A binding proteins binds
Where is the Poly A tail added?
nucleus, can be lengthened in cytosol in some cases
What signals termination to RNA pol?
a nuclease binds the uncapped 5’ end and degrades the trailing RNA, when it reaches RNA pol, it is terminated
What are the functions of the poly A tail?
- mRNA stability
- involved in transport of mature mRNA from nucleus to cytosol
- contributes to efficient translational initiation
What tRNA processing occurs in order to make sure tRNA is ready for protein synthesis?
- 16 nucleotide sequence at 5’ end is cleaved by RNAse P
- 14 nucleotide intron sequence in the anticodon loop is removed
- Uracil residues at the 3’ end are replaced by CCA sequence found in all mature tRNA
- many bases are converted to modified bases
Why is the change from Uracil to CCA on the 3’ end of tRNA important?
important step in quality control because enzymes only recognize tRNAs that are properly folded
What end of the tRNA does the amino acid attach?
3’
What reads the correct protein sequence in tRNA?
anticodon
How is the anticodon read?
5’ to 3’ or right to left
-codon is opposite
3’ UAC 5’ anticodon
5’ AUG 3’ codon
What does the S refer to in ribosomal subunits (45S)?
Svedberg units, sedimentation coefficients, which reflect both the shape and particle weight and are not additive
A single gene encodes what in ribosomal RNA? How are copies of this gene put together? Modifications? Processing?
- a large 45S precursor
- 250 to 300 copies of the gene are clustered together on afrocentric chromosomes
- some nucleotides of the rRNA precursor are chemically modified (methylation)
- Processed by cleavage
What is rRNA responsible for?
most of the catalytic activity of the ribosome
Where are ribosomal subunits assembled?
in the nucleolus by combination with over 80 ribosomal subunits
miRNA is involved in regulation of what?
up to 60% of protein encoding genes
What is miRNA transcribed by? What locations are miRNA found in the genome?
-RNA Pol II
- from introns or 3’ untranslated regions of pre-mRNA
- most processed from pre-miRNA- transcripts of 100-1000 nucleotides, some contain more than one miRNA
What is the role of miRNA?
- act as a guide sequence that brings nuclease into contact with target mRNA
- fine tune levels of protein made from the target mRNA under different conditions, not an all or none regulation
What are the steps to processing miRNA?
- miRNA sequence folds to form hairpin about 70 nucleotides long with imperfect base pairing
- RNase called Drosha that is specific for double stranded RNA cleaves or “crops” the hairpin to produce pre-miRNA
- pre-miRNA goes to cytosol
- RNase called Dicer, another double strand specific RNase, processes the pre-miRNA to produce a double stranded miRNA 21-23 nucleotides long
- one strand binds with an argonauts protein to form a mature RISC (RNA inducing silencing complex)
What happens if the complementarity between miRNA and its target is complete?
Argonaute slices the mRNA, removing the poly A tail and leaving it vulnerable to nucleases
- RISC complex is released and can bind to other copies of the mRNA
- less protein is translated
What happens if the match between miRNA and its target is less complete?
Argonauts does not slice the target, it inhibits translation and destabilizes the mRNA indirectly by moving it to processing bodies (P-bodies) in cytosol
-less protein is translated
What happens in P bodies?
mRNA is sequestered away from ribosomes and eventually degraded
What is a codon?
three mRNA nucleotides that encode an amino acid
What are three properties of the genetic code?
- Nonoverlapping, no punctuation - starts at AUG and reads 3 nucleotides at a time
- Specific - given codon always encodes the same amino acid
- Degenerate/redundant- an amino acid might be encoded by more than one codon
What makes the genetic code degenerate?
due to wobble in the binding between tRNA and mRNA codon
What base undergoes wobble movement in the tRNA anticodon?
5’ - far right
What strand does the mRNA sequence resemble? What is it complimentary to?
- coding
- template
What terminus of protein corresponds to the 5’ end of mRNA? 3’ end?
5’ = Amino (N)
3’ = Carboxy (C)
What are the 3 different point mutations?
- missense
- nonsense
- silent
What is a missense mutation?
one base is changed in the DNA sequence, which causes the new codon to encode a different amino acid, there is a change in the amino acid sequence
What is a nonsense mutation?
one base is changed in the DNA sequence, which causes the new codon to be a stop codon, there is a premature termination of translation
What is a silent mutation?
the new codon encodes the same amino acid, there is no change in the amino acid sequence
When could a silent mutation cause problems?
- could affect splicing by changing the marker for a splice site
- translation could be slower
- could affect protein folding
What is a frameshift mutation?
an insertion or deletion of any number of nucleotides not divisible by 3 changes the amino acid sequence, could cause premature termination
What is aminoacyl tRNA synthetase? How many are there? How many active sites does it have? What is its responsibility?
- enzyme that recognizes one amino acid and its corresponding tRNA
- 20 different ones for the different amino acids
- 2 active sites
- responsible for accuracy of translation
How does aminoacyl tRNA synthetase choose the right amino acid?
-two step double sieve editing mechanism
- the active site rejects amino acids that are larger than the correct one
- when tRNA binds the synthetase, it forces the amino acid-AMP into an editing pocket, the correct amino acid will not fit, but the smaller ones do and are hydrolyzed and released
- the correct amino acid is activated
What are the ribosome active sites in translation? where is the mRNA binding site?
A- active
P- peptide
E- Exit, mRNA binding site
What are the steps of initiation in translation?
- Initiator met-tRNA forms a complex with the active form of eIF-2-GTP
- Other eIFs bind separate the ribosomal subunits and bind the small, 40S subunit
- Initiator tRNA + eIF-2-GTP bind to the small ribosomal subunit to form an initiator complex
- mRNA bound by initiation factors, some that recognize the 5’ cap and poly A tail
- the initiator complex binds to the mRNA, and uses energy from ATP to move along the mRNA until it finds AUG, complimentary base pairs are formed between the start codon and the anticodon of tRNA
- large ribosomal subunit is added in a reaction that hydrolyzes GTP and releases initiation factors
What must happen to eIF-2-GDP before in cannot participate in initiation? What is its significance in initiation?
- in must be in active form, eIF-2-GTP
- it allows for global regulation of translation under certain conditions
What active site does initiator tRNA bind?
P site, leaving A site open, normally A site is first
Describe the three steps of elongation in translation?
Step 1-
- initiator met-tRNA complex occupies the P site
- tRNA containing an anticodon complementary to the next codon and charged with the correct amino acid enters the A site
- if correct, GTP is hydrolyzed and elongation factor is released along with GDP
Step 2-
- Peptidyl transferase (ribozyme), associated with large ribosomal subunit 60S, transfers the peptide chain from the tRNA in the P site to the amino group of the amino acid attached to the amino acid in the A site to form a peptide bond, condensation reaction
- conformational change in large subunit that shifts the two tRNAs to the E and P sites
Step 3-
- conformational changes moves the mRNA exactly three nucleotides through the ribosome and resets the ribosome so the A site is open
- Repeats adding one amino acid at a time
Where does the new tRNA enter?
A site
What enzyme makes a peptide bond? What type of reaction is it involved in?
- peptidyl transferase, activity of rRNA in large subunit
- condensation reaction, releases H2O
What is the significance of conformational changes in the ribosomal subunit?
- shift tRNAs to E and P sites
- move mRNA exactly three nucleotides
- reset ribosome so A site is open
Describe the process of termination in translation?
- when the codon in the A site is a termination codon, eRF-GTP (Euk release factor) binds to the A site
- protein is released from tRNA by peptidyl transferase in a reaction that involves the addition of water to the COO- terminus group of the last amino acid and the hydrolysis of GTP
- uncharged tRNA dissociates from the ribosome and ribosome dissociates from mRNA
What are the three stop codons?
UAA
UAG
UGA
What are the polyribosomes, polysomes?
consist of a single mRNA with multiple ribosome attached, translating at the same time
What proteins are translated more quickly?
ones that are more abundant
How long does the synthesis of proteins take?
20 seconds to several minutes
What do poly A binding proteins interact with?
initiation factors
