Transcription and Translation Flashcards
Where does genetic information usually flow?
- From DNA to DNA during its transmission from generation to generation
- From DNA to protein during its phenotypic expression in an organism
What steps are involved in the transfer of genetic info from DNA to protein?
- Transcription, the transfer of the genetic information from DNA to RNA
- Translation, the transfer of information from RNA to protein
- AKA the Central Dogma
Draw and label a diagram of the process of Central Dogma.
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
How does transcription and translation work in prokaryotes?
- The primary transcript is equivalent to the mRNA molecule
- The mRNA codons on the mRNA are translated into an amino acid sequence by the ribosomes
How does transcription and translation work in Eukaryotes?
- The primary transcript (premRNA) is a precursor to the mRNA
- The pre-mRNA is modified at both ends, and introns are removed to produce the mRNA
- After processing, the mRNA is exported to the cytoplasm for translation by ribosomes.
Study the diagrams of transcription and translation in prokaryotes and eukaryotes.
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
What are the general features of RNA synthesis?
Similar to DNA synthesis except
- The precursors are ribonucleotide triphosphates.
- Only one strand of DNA is used as a template.
- RNA chains can be initiated de novo (no primer required).
- The RNA molecule will be complementary to the DNA template strand and identical (except that uridine replaces thymidine) to the DNA nontemplate strand.
Which strand is the mRNA strand identical to?
- The non-template strand
- the strand that runs 5’ to 3’
Study the process of the transcription and translation of mRNA
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
What is RNA polymerase?
- RNA polymerase is an enzyme that is responsible for copying a DNA sequence into an RNA sequence, during the process of transcription.
What does RNA polymerase do?
- RNA synthesis is catalyzed by RNA polymerases and proceeds in the 5’ →3’ direction
What are some differences between RNA and DNA synthesis?
- Nucleotide sugar is ribose not 2-deoxyribose
- Uridine (base = uracil) replaces thymidine (base = thymine)
What are promoters?
- In genetics, a promoter is a region of DNA that leads to initiation of transcription of a particular gene.
How is RNA synthesis initiated?
- RNA polymerases bind specific nucleotide sequences called promoters, and helped by transcription factors initiate RNA synthesis at transcription start sites near the promoters
Where does RNA synthesis occur?
- RNA synthesis takes place within a locally unwound segment of DNA, sometimes called a transcription bubble, which is produced by RNA polymerase
- This allows a few nucleotides in the template strand to base-pair with the growing end of the RNA chain
What was on summary slide 1?
- In eukaryotes, genes are present in the nucleus, whereas polypeptides are synthesized in the cytoplasm.
- Messenger RNA molecules function as intermediaries that carry genetic information from DNA to the ribosomes, where proteins are synthesized.
- RNA synthesis, catalyzed by RNA polymerases, is similar to DNA synthesis in many respects.
- RNA synthesis occurs within a localized region of strand separation, and only one strand of DNA functions as a template for RNA synthesis.
What are the three stages of transcription in prokaryotes?
- Initiation of a new RNA chain
- Elongation of the chain
- Termination of transcription and release of the nascent RNA molecule
Study the diagram of RNA polymerase function and the diagram of Transcription in prokaryotes.
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
What is the difference between DNA polymerase and RNA polymerase?
- DNA polymerase is in replication
- RNA polymerase is in transcription
Where are the transcription regions in prokaryotes?
- Upstream: regions located toward the 5′end
- Downstream: regions located toward the 3′end
What are the numbering of transcription units?
- The transcript initiation site is +1
- Bases preceding the initiation site are given minus (–) prefixes and are referred to as upstream sequences.
- Bases following (relative to the direction of transcription) the initiation site are given plus (+) prefixes and are referred to as downstream sequences.
What is a consensus sequence?
- A sequence of DNA having similar structure and function in different organisms.
What is a recognition sequence?
- A DNA sequence to which a structural motif of a DNA binding domain exhibits binding specificity
What are the consensus & recognition sequences in an E. coli promoter?
- Consensus sequences: -10 sequence and -35 sequence
- Recognition sequence: -35 sequence
Why are the consensus and recognition sequences important?
- These DNA sequences are recognised by the transcription apparatus and are required for transcription to take place
What do the consensus and recognition sequences do?
- The -35 sequence is initially recognised and bound by sigma (σ ) subunit, used for orientation
- The -10 (AT-rich) facilitates the unwinding of the DNA, aka the “TATAA box”
Why is the “TATAA box” important?
- The -10 sequence is composed of T’s and A’s therefore it easiest to have a point of origin start here.
- It is where unwinding occurs
Study the diagram of the E.coli promoter.
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
Give an example of how DNA is transcribed into mRNA using the -10 and -35 sequences
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
When does termination occur?
- Termination of RNA chains occurs when RNA polymerase encounters a termination signal
What is Rho?
- A type of protein
What are the two termination signals in E.coli?
- Rho-dependent terminator —require a protein factor (ρ, or “Rho”), still being studied by scientists
- Rho-independent terminators—do not require ρ
Why are we only focusing on Rho-independent terminators?
- Rho-independent termination better understood due to the conserved nature of the DNA sequences
What is the structure of Rho-independent terminators?
- GC-rich region followed by 6 AT base pairs
- RNA chains with GC-rich regions form hairpin structures that impede movement of RNA polymerase
Study the diagram of the termination process of RNA.
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
What actions happen simultaneously in regard to mRNA?
- Since mRNA molecules are synthesized, translated, and degraded in the 5′to 3′direction, all three processes can occur simultaneously on the same RNA molecule
What was on summary slide 2?
- RNA synthesis occurs in three stages: (1) initiation, (2) elongation, and (3) termination.
- RNA polymerases—the enzymes that catalyze transcription—are complex multimeric proteins.
- Promoters are DNA sequences recognised by the transcription apparatus and are required for transcription to initiate and take place
- The covalent extension of RNA chains occurs within locally unwound segments of DNA.
- Chain elongation stops when RNA polymerase encounters a transcription-termination signal.
What does the codon AUG encode for?
- AUG encodes for the amino acid Methionine
What are the additional features of Eukaryotic transcription that prokaryotes don’t have?
- 7-methyl guanadine cap added to the 5ʹ of the primary transcript
- Poly(A) tail added to the 3ʹ of the primary transcript
- Removal of intron sequences
Study the diagram of Transcription and RNA Processing in Eukaryotes as well as the table for the three RNA polymerases
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
How many RNA polymerases do Eukaryotes have?
- RNA Polymerase I
- RNA Polymerase II
- RNA Polymerase III
What does RNA polymerase I do in Eukaryotes?
- RNA polymerase I: Synthesizes all but one rRNA. Located in the nucleolus (a protein structure within the nucleus)
What does RNA polymerase II do in Eukaryotes?
- RNA polymerase II: Responsible for the expression of genes that encode for long transcripts, many of which are translated into protein.
What does RNA polymerase III do in Eukaryotes?
- RNA polymerase III: Synthesizes many small RNAs such as the tRNAs, 5s rRNA and siRNA (a more recent discovery) important for gene regulation.
What is the difference between a regulatory and a core promoter?
- A core promoter is that portion of the proximal promoter that contains the transcription start sites
- A regulatory promoter is the binding site for the basal transcriptional apparatus
Why is the RNA polymerase II promoter important?
- Locally unwound segment of DNA are required to initiate transcription
- This involves interaction of transcription factors with specific promoter sequences
- The promoter consists of short conserved elements upstream of the transcription start point (+1)
What “boxes” are required for transcription? Also, study the diagram for the structure of these boxes.
- TATA box: Element closest to transcription start. Plays important role in positioning the transcription startpoint and helps unwinding
- CAAT box: Influences efficiency
- GC & Octamer box: Often present, they influence efficiency
https: //docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
Why are the CAAT, GC and Octamer boxes important?
- They bind transcriptional
activator proteins
What are the three types of terminators?
- UGA
- UAG
- UAA
What is a 7-MG cap
- AKA “7-Methyl Guanosine”
- The cap contains a guanosine that is methylated at the position 7
- Cap added after the chain is ~30 nucleotides long
What is the purpose of a 7-MG cap
The cap has two functions:
- Prevents degradation of the RNA transcript from RNases
- Is recognised by the proteins that initiate translation of the RNA transcript into a protein
What is the 3’Poly (A) tail?
- Polyadenylation is the addition of a poly tail to a mRNA. The poly tail consists of multiple A’s. In eukaryotes, polyadenylation is part of the process that produces mature mRNA for translation.
Why is the Poly(A) tail significant for transcription?
- Poly(A) tail enhances stability of mRNAs and plays important role in their transport from nucleus to cytoplasm
How is the Poly(A) Tail added?
- Once the polyadenylation sequence (AAUAAA) and the GU rich sequences have been transcribed a protein complex containing an endonuclease and poly(A) polymerase binds to these sequences and cleaves 11bp downstream of the AAUAAA
- After cleavage the polyA tail is added (up to 200 bps long) by the polyApolymerase
Why are Intron’s taken out of the mRNA strand?
- Most eukaryotic genes contain noncoding sequences called introns that interrupt the coding sequences, aka exons
- The introns are excised from the RNA transcripts prior to their transport to the cytoplasm
Provide a summary of introns.
- Introns (or intervening sequences) are noncoding sequences located between coding sequences.
- Introns are removed from the pre-mRNA and are not present in the mRNA.
- Exons (both coding and noncoding sequences) are composed of the sequences that remain in the mature mRNA after splicing.
- Introns are variable in size and may be very large.
Which enzyme does splicing?
- Endonuclease
Why is the Excision of Intron Sequences important?
- The third modification of eukaryotic RNA transcripts is the removal of introns.
- This occurs within the nucleus and that the removal of introns allows different proteins to be synthesized by creating multiple transcripts – increasing complexity phenotypically without the need for an increase in the genotype.
Study the diagrams for the 7-MG cap, the 3’ Poly(A) tail and the Excision of Intron sequences.
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
What is on summary slide 3?
- Three different RNA polymerases are present in eukaryotes, and each polymerase transcribes a distinct set of genes.
- Eukaryotic gene transcripts usually undergo three major modifications:
(1) the addition of 7-methyl guanosine caps to 5’ termini,
(2) The addition of poly(A) tails to 3’ ends, and
(3) The excision of noncoding intron sequences.
What does translation involve?
- Translation involves three types of RNA, all of which are transcribed from DNA templates (chromosomal genes)
- In addition to mRNAs, three to five RNA molecules (rRNA) are present as part of the structure of each ribosome
Why are tRNA important?
- 40 to 60 small RNA molecules (tRNA) function as adaptors by mediating the incorporation of the proper amino acids into polypeptides in response to specific nucleotide sequences in mRNAs
Where does translation occur?
- Translation occurs on ribosomes, which are complex macromolecular structures located in the cytoplasm
What is aminoacyl-tRNA
synthetases
- The amino acids are attached to the correct tRNA molecules by a set of activating enzymes called aminoacyl-tRNA synthetases
Study the diagram of the process for Translation
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
What is rRNA?
- Ribosomal RNA
- rRNA are transcribed from a DNA template
- rRNA gene transcript is a 30S precursor, which undergoes endonucleolytic cleavages to produce the 5S, 16S, and 23S rRNAs plus one 4S tRNA molecule
What are prokaryotic ribosomes and how are they composed?
- Ribosomes: half protein and half RNA
- Each subunit contains large, folded RNA molecule on which the ribosomal proteins assemble
- 30S ribosomal subunit contains a 16S RNA molecule plus 21 polypeptides. The 50S subunit contains two RNA molecules (5S and 23S) plus 31 polypeptides
What are tRNA?
- Transfer RNA
- tRNAs are derived from larger precursor transcripts in the same way rRNA
- tRNA molecules contain a triplet nucleotide sequence, the anticodon, which is complementary to and base pairs with the codon sequence in mRNA during translation
- There are one to four tRNAs for each of the 20 amino acids
Why are tRNA important?
- Amino acids are attached to the tRNAs by high-energy (very reactive) bonds between the carboxyl groups of the amino acids and the 3ʹ hydroxyl termini of the tRNAs
- The aminoacyl~tRNAs are the substrates for polypeptide synthesis on ribosomes, with each activated tRNA recognizing the correct mRNA codon and presenting the amino acid in a steric configuration (3D-structure) that facilitates peptide bond formation
How many binding sites are there on each ribosome and what are they?
- Three tRNA binding sites on each ribosome
- The A or aminoacyl site binds the incoming aminoacyl-tRNA, the tRNA carrying the next amino acid to be added to the growing polypeptide chain
- The P or peptidyl site binds the tRNA to which the growing polypeptide is attached
- The E or exit site binds the departing uncharged tRNA
Where in the ribosome are the tRNA binding sites?
- An mRNA molecule is attached to the 30S subunit → contributes specificity to the tRNA-binding sites located largely on the 50S subunit of the ribosome
Where does the initiation of translation in E. coli occur?
- Formation of the 30S subunit/mRNA complex depends, in part, on base pairing between two nucleotide sequences.
- They include a nucleotide sequence near the 3ʹ-end of the 16S rRNA and a sequence (Shine-Dalgarno sequence) near the 5ʹ-end of the mRNA molecule
- This is usually 7 nucleotides upstream of AUG (start codon)
Study the diagrams for Prokaryotic Ribosome synthesis, tRNA , Translation Complex and the initiation of Translation
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
When does polypeptide chain termination occur?
- Polypeptide chain termination occurs when a chain-termination codon (stop codon) on the mRNA enters the A site of the ribosome
- The stop codons are UAA, UAG, and UGA
What happens during polypeptide chain termination?
- When a stop codon is encountered, a release factor (RF-1) binds to the A site
- A water molecule is added to the carboxyl terminus of the nascent polypeptide, causing termination.
What are some properties of the genetic code?
- Composed of nucleotide triplets
- Non-overlapping
- Comma free
- Degenerate
- Ordered (similar amino acids specified by related codons)
- Contains start and stop codons
- Nearly universal
What does “Degenerate” mean in genetics?
- Having more than one codon that may code for the same amino acid.
How does Degenerate Genetic code work?
- 20 amino acids = at least 20 different codons using four bases available in mRNA
- Two bases per codon = only 42 or 16 possible codons (too few!)
- Three bases per codon = 43 or 64 possible codons (too many?)
- All amino acids except methionine and tryptophan are specified by more than one codon
Give examples of Degenerate genetic code.
- leucine, serine, and arginine—are each specified by six different codons
- Isoleucine has three codons
- Other amino acids each have either two or four codons
What is Degeneracy?
- The occurrence of more than one codon per amino acid is called degeneracy (not random)
What types of degeneracy are there?
Degeneracy is primarily of two types:
- Partial degeneracy: 3rd base either two pyrimidines (U or C) or two purines (A or G). Changing the third base from a purine to a pyrimidine, or vice versa, will change the amino acid specified by the codon
- Complete degeneracy: Any of the four bases may be present at the third position in the codon, and the codon will still specify the same amino acid.
Study the diagrams for polypeptide chain termination and degenerate genetic code
https://docs.google.com/document/d/1Nzo4FTzXCbwOZjpoc_J_4IF3gsOXPcoyC2BowELmx0U/edit?usp=sharing
How does a “wobble” occur?
- Hydrogen bonding between bases in the anticodons of tRNAs and the codons of mRNAs follows strict base pairing rules only for the first two bases of the codon
- The base-pairing involving the third base of the codon is less stringent, allowing what is called wobble at this site
What is the significance of the wobble base?
- Mutations at this site have less impact than the first two positions → decreasing the effect of mutations
How does a wobble base decrease the effect of mutations?
- Often amino acids with similar physical properties share similar codon sequences and often differ by a single base, thus if a coding mutation is created then the substituted amino acid is more likely to share similar physical properties and ultimately minimise the effect of protein sequence/ structure/function
What is Inosine?
- Several tRNAs contain the base inosine. Inosine is produced by a post-transcriptional modification of adenosine
- Wobble hypothesis predicted that when inosine is present at the 5ʹ-end of an anticodon (the wobble position) it would base-pair with uracil, cytosine, or adenine in the codon
- Experimental evidence: Purified alanyl-tRNA containing inosine (I) at the 5ʹ-position of the anticodon binds to ribosomes activated with GCU, GCC, or GCA trinucleotides
What was on the last summary slide?
- Each of the 20 amino acids in proteins is specified by one or more nucleotide triplets in mRNA
- Of the 64 possible triplets, given the four bases in mRNA, 61 specify amino acids and 3 signal chain termination
- The code is nonoverlapping, with each nucleotide part of a single codon, degenerate, with most amino acids specified by two to four codons, and ordered, with similar amino acids specified by related codons
- The genetic code is nearly universal; with minor exceptions, the 64 triplets have the same meaning in all organisms
- The wobble hypothesis explains how a single tRNA can respond to two or more codons
