The Genetic Code and Transcription

Vocabulary:

• Central Dogma of Molecular Biology: Describes the unidirectional flow of genetic information from DNA to RNA to protein, transcription copies DNA into an mRNA sequence, translated in ribosomes using tRNA into an amino acid sequence

• Messenger RNA (mRNA): The complement of a template strand, acts as a messenger molecule being transported out of the nucleus, will associate with ribosomes where it is decoded and translated into a protein

• Triplet code: “words” for genetic code, consists of 3 ribonucleotide letters, 64 unique arrangements that only specify 20 amino acids

• Codon: Consists of one triplet code, has 3 ribonucleotides

• Unambiguity: A property of genetic code in translation, each codon only corresponds to one amino acid

• Degeneracy: A property of genetic code in translation, a given amino acid can correspond to multiple codons

• Commaless: A property of translation, once there is a start signal codons are read with no break, until there is a stop signal

• Nonoverlapping: A property of translation: a single ribonucleotide within mRNA is only part of one triplet

• Colinear: A property of genetic code in translation, the sequence of codons determines the order of the amino acids in the encoded protein

• Universal: A property with only one exception in translation, a single coding dictionary is used by all organisms including viruses, prokaryotes, archaea, and eukaryotes

• Methionine (MET): An amino acid encoded by the AUG triplet, can initiate a polypeptide chain

• Termination signal: A triplet that instead of encoding an amino acid will stop polypeptide synthesis, UAA UAG of UGA

• Inosine: A base in tRNA that can match with A, U, or C

• Reading frame: Where mRNA will start reading 5’→3’ mRNA, based on a contiguous sequence of nucleotides, though disrupted by frameshift mutation will be reestablished with 3 insertions/deletions

• Frameshift mutation: A mutation that will change the reading frame, caused by an insertion or deletion of a nucleotide

• Sydney Brenner: Coined the term ‘codon’ in the 1960s, theorized mathematically that it was a triplet

• Nirenberg and Matthaei: In 1961 deciphered the first codon UUU, encoding phenylalanine and showed that RNA controlled the production of proteins. Extracted the cellular machinery from E. coli responsible for protein production and demonstrated that proteins could be made when to intact living cell was present, used cell-free protein synthesis to link specific coding sequences to specific amino acids

• Cell-Free Protein Synthesis: Done in cell lysate, which had all essential factors for protein synthesis without organelles or a cell membrane, uses the enzyme polynucleotide phosphorylase to synthesize RNA templates for mRNA, generates random RNA

• RNA Homopolymer codes: RNA molecules that only contained a single type of nucleotide

• tRNA: A kind of RNA used in translation, contains an anti-codon and is attached to an amino acid

• Anti-codon: A complementary sequence to mRNA that corresponds to a single amino acid

• Nirenberg and Leder: Looked into how specific sequence assignment for triplet codons was made possible by triplet binding assay, observed that ribosomes could bind sequences as short as 3 ribonucleotides. Radioactively labeled amino acids and added them to a cell-free system, enzymes in the lysate attached the radioactive amino acids to tRNAs, used knowledge of codon composition to narrow down which amino acid was each specific triplet. Concluded code was degenerate and unambiguous

• Triplet binding assay: The experiment that led to the sequencing of every amino acid. Knew the compositions of each polyribonucleotide, and could synthesize tRNA with unique sequences. Radioactively labelled the amino acids, and filtered them by size which excluded tRNAs that were bound to codons

• Wobble hypothesis: The initial two ribonucleotides of the triplet codes are often more critical than the third. This allows for degeneracy using the third positioned ribonucleotide in tRNA, it is less spatially constrained and doesn’t need to adhere as strictly to established base-pairing rules

• N-formylmethionine (fmet): A modified form of methionine found in bacteria

• Nonsense mutations: A mutation that can produce a stop codon internally prematurely, terminating translation and producing only a partial polypeptide

• Mitochondrial DNA (mtDNA): Has many exception to universal genetic code, UGA typically specifies termination but encodes tryptophan in yeast and humans. AUA normally encodes isoleucine but encodes internal insertion of methionine

• Overlapping genes: When a single mRNA strand may have multiple initiation points, which creates different reading frames and may specify more than one polypeptide. This is common in viruses

• Open reading frame (ORF): Happens with overlapping genes, a series of triplet codons will specify amino acids to make a polypeptide and initiation at different AUG positions within the frame will results in distinct polypeptides

• Template strand: The strand in DNA that is actually transcribed into mRNA in the 5’→3’ direction

• Coding strand: The strand of DNA that is not transcribed

• Promoter: A regulatory sequence upstream of the 5’ end where RNA is transcribed, part of DNA, non-coding but where the transcription machinery binds, identifies where a gene starts and which strand to use as the template, giving it orientation. Not transcribed itself

• RNA-coding region: The part of DNA that is transcribed into mRNA, will likely contain protein coding and protein non coding sequences

• Terminator: Part of DNA, ends transcription, consists of a regulatory sequence, part of the mRNA transcript created

• RNA Polymerase: An enzyme that directs the synthesis of RNA from a DNA template, doesn’t use a primer and its nucleotides contain ribose not deoxyribose

• Bacterial RNA Polymerase: Contains 5 subunits, 2 alpha, and one of each beta, beta’, and omega. The holoenzyme contains an additional sigma factor, which can bind to a promoter and initiate transcription. Different sigma subunits recognize different subsets of promoters, this mechanism is used to regulate gene expression under varying environmental conditions like heat/starvation. The sigma subunit dissociates while elongation proceeds with just the core enzyme (50/sec at 310 K)

• Initiation: A mechanism of transcription, it is the assembly of apparatus at the promoter and the start of RNA synthesis

• Transcription start site (TSS): Where transcription begins, indicated numerically as position +1

• Elongation: Mechanism of transcription, this is the reading of the template DNA and the formation of a polynucleotide RNA molecule

• Termination: A mechanism of transcription, this is identification of the transcription stop site, dissociation of the transcriptional apparatus, and separation of the new RNA molecule from the DNA template

• Consensus sequences: DNA sequences homologous in different genes of the same organism

• Cis-acting elements: Consensus sequences located neat the region to be transcribed

• Trans-acting factors: Proteins that influence gene expression by binding to cis-acting elements. One example is sigma factor in bacterial RNAP

• Rho-independent termination: An enzyme traverses the entire gene until a termination sequence is encountered, self-complementary GC rich sequences form a hairpin which causes RNAP to stall adjacent to the poly U track, which as relatively weak interactions causing the RNAP to dissociate and release the transcript

• Rho dependent termination: Termination in bacteria that relies on the rho dependent factor, which recognizes a binding site (rut) as soon as it is transcribed. Rho has helicase activity, and has a sequence to bind and cause the polymerase to stall and create a hairpin

• Rho utilization site (rut): Where Rho binds to initiate the process of transcription ending

• Rho: Binds to rut, will break hydrogen bonds and physically disassemble RNA polymerase

• Hairpin secondary structure: Part of termination in bacteria, part of the newly formed transcript folds back in on itself forming this

• Chromatin remodeling: The process of chromatin uncoiling to make DNA accessible to RNAP

• RNA Polymerase I: Produces RNA, located in the nucleolus

• RNA Polymerase II: Produces mRNA and snRNA, in the nucleoplasm, dependent of cis-acting elements and trans-acting transcription factors, binds to where the core-promoter is

• RNA Polymerase III: Produces 5SrRNA and tRNA, located in the nucleoplasm

• Proximal-promoter elements: Consist of enhancers and silencers, found upstream, within, or downstream of a gene, used in gene regulation and expression

• Enhancer: Increases transcription levels

• Silencer: Decreases transcription levels

• General transcription factors (GTPs): Required by all RNAP II for transcription

• Transcription activators and repressors: Influence efficiency or rate of RNAP II transcription initiation

• TATA box: A core promoter element, binds TBP (TATA-binding protein) of transcription factor TFIID to determine the transcription start site

• Pre-initiation complex: Formed by TBP binding to the TATA box and other transcription factors like TFDII binding allowing RNAP II to bind to it

• DNA-RNA duplex: Part of RNA Pol II, bent at a right angle which positions the 3’ end of the RNA at the active site of the enzyme, allowing nucleotides to be added to the 3’ end

• Abortive transcription: Transcription within RNA Pol II that is under 11 nucleotide pairs, due to it being initially unstable

• Polyadenylation signal sequence: Signals termination in eukaryotes once detecting AAUAAA, the transcript is cleaves 10-35 base pairs further downstream

• Rat1: An enzyme in eukaryotes for termination, works 5’→3’ with exonuclease activity. Attaches to a cleaved 5’ end of the RNA, moves down, and reaches the polymerase enzyme

• pre-mRNA: The transcript directly out of transcription, requires further processing. It is much longer and needs to be transported out of the nucleus and into the cytoplasm. Will get the addition of a 5’ cap (7-mg cap), addition of 3’ tail (poly-A tail), and have excision of introns and undergo slicing

• Poly-A polymerase: Synthesizes a stretch of 250 adenylic acid residues at the 3’ end, attaches after the polyadenylation signal sequence (AAUAAA)

• Poly A binding protein: Binds to the tail of pre-mRNA to protect it

• Introns: Regions of initial RNA transcription that aren’t expressed in amino acid sequences of the protein

• Exons: Sequences retained and expressed, mostly present in only eukaryotes

• Heteroduplexes: Introns present in DNA but not mRNA loop out

• Untranslated regions (UTRs): RNA regions that will not be translated but regions that have other functions like binding to protein-making machinery

• Splicing: The process that removes introns and joins exons together in mature mRNA

• Heterogenous RNA (hnRNA): mRNA that contains all introns and exons, pre-splicing

• Alternative RNA splicing: Allows for different segments to be treated as exons during RNA processing, the presence of introns allows a single gene to encode more than one kind of polypeptide

• Self-splicing RNAs: The intron is the source of enzymatic activity and removes itself through self-excision, requires ribozymes

• Ribozyme: An enzymatic RNA

• Spliceosome: Where pre-mRNA introns are spliced out, uses reactions that involve formation of lariat structure, splice donor and acceptor sites, and branch point sequences

• Prinbow box: A bacterial consensus sequence, example is TTGACA

In eukaryotes transcription occurs in the nucleus, in prokaryotes it is in the free cell. mRNA leaves the nuclease for translation

Transcription is initiated at RNA polymerase II promoters when TFIID and TBP transcription factors bind to the TATA box, followed by the binding of other general transcription factors, allowing RNAP II to bind to TFIID

The DNA double helix will enter RNA Pol II through a grove and unwind. Cleaving will destabilize RNAPII, opening up the clamp and releasing DNA and RNA