Transcription + Translation Flashcards
What is trascription?
ribonucleic acid synthesis from DNA template
What is the primary and secondary structure of RNA?
primary: existing in single strands
secondary: folds back upon itself in regions where complimentary base pairing is possible
What are the three types of RNA and how much they make up the RNA in a cell’s cytosol?
mRNA: 1-2% cytosol RNA
tRNA: 15% cytosol
rRNA: 85% cytosol RNA
What does the stucture of RNA affect and why?
affects longevity as mRNA are only in primary structure which degrades easily and tRNAs/rRNAs are in secondary structure which prevent ribonuclease attack
What are the features in a bacterial gene for transcription?
- Promoter: -35 (recognized by sigma factors) and -10 boxes (pribnow box)
- Leader sequence: transcribed into mRNA but not translated
- Coding region: transcribed and translated into polypeptide
- Trailer: preps mRNA to release template strand; transcribed into mRNA but not translated
- Terminator: RNA polymerase releases the template strand
What is RNA polymerase’s structure and function?
Structure: Large multimeric enzyme with 2 alpha (a) subunits, beta subunit (B), beta’ subunit (B’), and omega (w)
Function: responsible for RNA synthesis
What is the sigma factor?
Controls binding of RNA polymerase to promoter and detaches once first few RNA nucleotides have been joined together; different sigma factors initiate binding of RNA polymerases to different promoters.
What are the steps of transcription initiation?
- sigma factor recognizes -35 box on promoter and binds first, then binds to -10 box forming a closed complex
- Complex opens with the melting of the -10 region by RNA polymerase and 16-20bp are unwound
- Sigma factor dissociates and RNA polymerase core enzyme responsible for elongation of RNA transcript
How does transcription elongation work?
- Core enzyme reads strand in 3’-5’ direction and adds rNTPs (ribonucleoside triphosphates)
- As nucleotides are added, a temporary double stranded RNA/DNA hybrid is formed, but the new RNA transcript separate from template
- Single stranded DNA rewinds to reform double stranded DNA helical structure
How does transcription termination work?
Inverted repeats at the end of gene sequence in the coding strand allow for hairpin/stemloop formation at the end of the RNA
What is the purpose of the hairpin structure at the end of an RNA transcript?
Secondary structure presents physical obstruction to cause RNA polymerase to stall and dissociate via two mechanisms of rho dependent termination
How does the poly-U mechanism of termination work?
A rich region present after inverted repeats in DNA allow for poly-U in RNA that causes destabilization of RNA/DNA hybrid in the open complex and releases RNA polymerase
How does rut site mechanism of termination work?
- rut site is activated when transcribed into RNA and will be bound by multi-rho hexameric protein that assembles around the RNA transcript.
- Multi-Rho protein uses ATP hydrolysis to act as helicase, knocking off RNA polymerase as it is stalled by the hairpin structure.
What are transcriptional units and examples?
Segments of DNA that are transcribed into a single RNA molecule bounded by their initiation and termination sites. (e.g. monocistronic transcripts, polycistronic transcripts, tRNAs, rRNAs, non-coding RNAs
What is the difference between a monocistronic and polycistronic transcript?
mono: RNA transcribed from a single gene with is own promoter and terminator
poly: RNA transcribed from two or more genes (co-transcribed genes)
What is an operon structure?
Two or more genes (co-transcribed genes) flanked by one promoter and one terminator.
What are some features of an operon?
- polycistronic mRNA transcribed contains multiple ribosome binding sites upstream of coding sequences
- One promoter may form a polycistronic RNA that may be processed to form multiple, individual RNA molecules by ribonucleases.
How are tRNA’s synthesized?
Transcribed as large linear precursor tRNA with some regions of complementary binding. Then, they are further processed by exonucleases and endonucleases to produce extensive secondary structures via base-paired regions
What is the result of post-transcriptional modification of purine and pyrimidine bases of tRNA?
Dihydrouridine (D): fully saturated pyrimidine ring
Pseudouridine (ψ): ribose joined to carbon-5 instead of nitrogen-1
Ribothymidine (T): methyl is added to carbon 5 of uridine
Methylguanosine (mG)
Modified purines (Y)
Where are the genes encoding tRNA’s located?
- May be present in rRNA operon
- May form operon consisting of 2-7 different tRNA genes
- May be monocistronic
How many amino acids are there?
20 amino acids and 2 unusual amino acids.
In what direction is mRNA read?
mRNA is read by translation machinery in the 5’-3’ direction in sets of 3 (codon)
What does it mean when the genetic code is degenerate?
There is more than one codon that codes for a single amino acid to minimize mutations.
What are the start and stop codons?
Start: AUG (methionine)
Stop: UAA, UAG, UGA
How many mRNA codons and tRNA anticodons are there?
Codons: 61 AA coding, 3 stop
Anticodons: 20 (1 per AA)
What is the wobble concept?
Flexible base pairing (due to base modifications) between the 3rd position of the mRNA codon and the 1st position of the tRNA anticodon.
What are the two important components of the tRNA structure?
- Acceptor domain: 3’ extension (5’-CCA-3’) where amino acid is attached
- Anti-codon loop: contains 3 bases of anti codon
What is the CCA-adding enzyme?
Enzyme that uses CTP and ATP as substrates to sequentially add amino acids to the A of CCA-3’
What does an aminoacyl-tRNA synthestase do?
Specific synthestase links correct amino acid to tRNA upon recogniztion of specific contacts (D loop, anticodon, parts of acceptor stem) via activation and charging
How does an amino acid become activated to be added to tRNA?
Amino acid reacts with ATP to form aminoacyl-AMP and pyrophosphate (PPi). Aminoacyl-AMP will remain bound to the synthetase until collision with the appropriate tRNA molecule
How does an activated amino acid form a charged tRNA?
Aminocyl-AMP reacts with CCA stem of tRNA and forms aminoacyl-tRNA + AMP.
What is the shine-Dalgarno sequence?
Sequence on mRNA for ribosome binding
What is the leader sequence?
5’ end of mRNA transcript, contains Shine-Dalgarno sequence which allows proper alignment of ribosome at starting codon in the coding sequence
What is the coding sequence?
Area between translational start and stop codons, including those codons, generally beginning with AUG start codon that encodes a chemically modified methionine (N-formylmethionine)
What is the trailer sequence?
Contains a sequence that stabilizes mRNA by impeding degradation due to its secondary structure
What’s the differnece between a -X and +X frame shift?
-X: reading frame is shifted X amount of bases to the left
+X: reading frame is shifted X amount of bases to the right
What are the 3 stages of translation?
Initiation, elongation, and termination
What are the two protein/rRNA subunits and their makeup?
50S subunit: 5S rRNA, 23S rRNA, 31 proteins
30S subunit: 16S rRNA, 21 proteins
What does the initiation complex require?
- mRNA transcript
- 30S and 50S subunits
- Initiation factors (IF-1, 2, and 3)
- Initial tRNA holding N-formylmethionine (fMet-tRNA)
- GTP for activation
What is the ribosomal binding site and where is it found?
Positions ribosome at start codon AUG as consensus sequence (UAAGGAGGU) complimentary to sequence within 16S rRNA.
What are the steps of initiation of translation?
- 16S rRNA of small subunit hydrogen binds to complimentary sequence at ribosomal binding site
2.transformylase modifies Met of tRNA^fMet by adding a formyl group to amino group, and is placed in P site
- 50S subunit is added
What are the 3 sites in the ribosome complex?
A (acceptor): entry of subsequent charged AA-tRNAs
P (peptide): fMEt-tRNA initiation codon and peptide bond formation
E (exit): release of used tRNAs
What is the process of mRNA binding to 30S and 50S subunit?
- 16S rRNA of small subunit hydrogen binds to complimentary sequence at ribosomal binding site
2.transformylase modifies Met of tRNA^fMet by adding a formyl group to amino group, and is placed in P site
- IF-3 prevents 30S subunit from binding to 50S subunit prematurely and IF-1 prevents inadvertent loading of N-formylmethionine in A site
- 3’ end of 16S rRNA is complementary to the Shine-Dalgarno sequence aligning start codon with P site
- IF-2 binds GTP then binds fMet-tRNA and guides it to P site
- IF-1 and IF-3 are released when tRNA is aligned with the start codon
- IF-2 cleaves GTP finalizing locking fMet-tRNA in place
- Binding of 50S subunit completes ribosome complex.
What are the steps of translation?
- Binding of aminoacyl-tRNA to the A site
- Peptidyltransferase (23S rRNA ribozyme) catalyzes peptide bond formation between COO- end of P site polypeptide and NH3+ end of A site amino acid
- P site is transferred onto amino acid in the A site
What is a polysome?
complex of several ribosomes translating a single mRNA molecule to increase the speed and efficiency of translation
What is a stalled ribosome?
Ribosome that is trapped on a defective mRNA that lacks a stop codon. Freed via trans-translation.
What are the steps of trans translation?
- tmRNA mimics tRNA (carries alanine) and mRNA (short sequence + stop codon)
- tmRNA collides with stalled ribosome and binds alongside defective mRNA
- protein synthesis proceeds by adding alanine to the chain and then reading the short tmRNA sequence
- Stop codon is reached and protein and ribosomes dissociate
- Added amino acid sequence signals protease to degrade resulting protein
What are DnaK and DnaJ?
ATP-dependent chaperone enzymes that bind to newly formed polypeptides and prevent from folding too quickly
What are GroEL and GroES?
muti-subunit chaperone protein complexes that use ATP to correct and complete the folding of improperly folded proteins.
What are the 2 unusual amino acids?
selenocysteine and pyrrolysine
How is selenocysteine synthesized and encoded?
- tRNA^Sec is aminoacylated with serine by seryl-tRNA synthetase (SerRS)
- Serine is then converted to selenocysteine by selenocysteine synthase
- Encoded by stop codon UGA followed by selenocysteine insertion sequence element (SECIS)
How is pyrrolysine encoded?
Encoded by UAG and inserted by pyrrolysine sequence element.
