13: The Genetic Code and Transcription

Eukaryotic mRNAs require processing to produce mature mRNAs

-Addition of a 5′ cap -Addition of a 3′ tail -Excision of introns

The genetic code is

-Unambiguous: each triplet (codon) specifies only a single amino acid -Degenerate: a given amino acid can be specified by more than one triplet codon -Commaless: codons read one after another until a stop codon is reached -Nonoverlappping: any single nucleotide with the mRNA is part of only on triplet -Nearly universal: a single coding dictionary is used by all viruses, prokaryotes, archaea, and eukaryotes

Enhancers and Silencers

-can be upstream, within, or downstream of the gene -can modulate transcription from a distance -act to increase or decrease transcription in response to cell's requirement for a gene product or at a particular time during development or place within an organism

chromatin remodeling

Eukaryotic transcription requires chromatin to become uncoiled, making the DNA accessible to RNA polymerase and other regulatory proteins

Posttranscriotional RNA processing

Heterogeneous nuclear RNA (hnRNA) posttranscriptionally processed before it can leave the nucleus -Addition of a 5' cap that protects from nuclease attack and may be involved in the transport of the transcript across the nucleus -poly-A tail added to aid transport to cytoplasm -Introns are removed by splicing

Encountering termination nucleotide sequence

In bacteria this termination is transcribed into RNA and causes the newly formed transcript to fold back on itself, forming what is called a hairpin structure held together by hydrogen bonds. -In some cases, termination depends on the rho () termination factor

Transcription

RNA synthesized on DNA template -The genetic information stored in DNA is transferred to RNA, which serves as the intermediate molecule between DNA and proteins *The synthesis of an RNA transcript from a DNA template is called: Transcription

Influence the efficiency or rate of transcription initiation

Regulatory DNA sequences Proximal-promoter elements Enhancers Silencers

Translation initiation

Ribosomes are attached to partially transcribed mRNA molecules and initiate translation -Polyribosomes have been observed in both prokaryotes and eukaryotes

There are two main types of RNA editing prior to translation

Substitution editing: the identities of individual nucleotide bases are altered; prevalent in mitochondria and chloroplast RNA derived in plants Insertion/deletion editing: nucleotides added/deleted from the total number of bases

Termination codons

Three codons (UAG, UAA, and UGA) -do not code for any amino acid *The 3 stop codons are: UAA, UAG, and UGA

Promoter

Transcription begins with template binding by RNA polymerase at a promoter -The σ subunit is responsible for promoter recognition (initiation of transcription) -In addition to promoters, enhancers and silencers also control transcription regulation *The subunit of an E.coli RNA Polymerase that recognize the promoter sequence in the DNA are/is: σ subunit

TATA box

core promoter element that binds the TATA-binding protein (TBP) of transcription factor TFIID and determines the start site of transcription

Triplet Binding assay

developed by Nirenberg and Leder to determine other specific codon assignments -In this technique, ribosomes bind to a single codon of three nucleotides, and the complementary amino acid-charged tRNA will be able to bind

RNA Polymerase

directs the synthesis of RNA using a DNA template -No primer is required for initiation -uses ribonucleotides instead of deoxyribonucleotides.

open reading frame, ORF

in viruses, overlapping genes -in which initiation at different AUG positions out of frame with one another leads to distinct polypeptides

N-formylmethionine (fmet)

initial amino acid incorporated into all proteins is a modified form of methionine -AUG is the only codon to encode for methionine -When AUG appears internally in mRNA, an unformylated methionine is inserted into the protein

Triplet codons

once transferred to RNA, the genetic code exists as triplet codons, which are sets of 3 nucleotides -each nucleotide is one of 4 kinds of ribonucleotides composing RNA -the triplet code provides 64 codons to specify the 20 amino acids *all of the following were used to determine at least some of the codons (with respect to the amino acids they called for) in the genetic code: -Homopolymers Templates -Repeating Copolymers -Mixed Copolymers -Triplet binding assays

François Jacob and Jacques Monod

postulated existence of mRNA -First added RNA Homopolymers (RNA nucleotides with only one type of ribonucleotide) to the in vitro translation system to decipher which amino acids were encoded by the first few codons based on which amino acids were incorporated into the polypeptide -Then added RNA Heteropolymers (two or more different ribonucleosides) were used to decipher more codons employing the same method

Wobble hypothesis

predicts that the initial two ribonucleotides of triplet codes are often more critical than the third.

Crick, Barnett, Brenner, and Watts-Tobin

provided the first solid evidence for a triplet code -triplet nature of the code was revealed by Frameshift Mutations

Introns (intervening sequences)

regions of the initial RNA transcript that are not expressed in the amino acid sequence of the protein -removed by splicing by the Spliceosome (reaction involving formation of a lariat structure) -exons joined together in the mature mRNA

RNA polymerase II (RNP II)

responsible for a wide range of genes in eukaryotes -RNP II promoters have a core promoter element and promoter that determine where RNP II binds to the DNA and where it begins copying the DNA into RNA

transcription factors (TFs)

scan and bind to DNA for recognition by eukaryotic DNA Polymerases Two broad categories of transcription factors that facilitate RNP II binding and initiation of transcription -General TFs: are absolutely required for all RNP II-mediated transcription Transcription Activators and Repressors: influence the efficiency or the rate of RNP II transcription initiation

Transcription start site

site where transcription begins -the DNA double helix is unwound to make the template strand accessible to the action of RNA polymerase

Analysis of RNA produced immediately after bacteriophage infection of E. coli shows that

the base composition of the newly synthesized RNA resembles that of the phage DNA and not that of the bacterial host -This suggests that RNA synthesis may be a preliminary step in protein synthesis

Long RNAs with di-, tri-, and tetranucleotide repeats

used for in vitro translation to determine more codon assignments

degenerate

with many amino acids specified by more than one codon -Only tryptophan and methionine are encoded by a single codon

Consensus sequences

• 5'-TTGACA-3', positioned at -35 • 5'-TATTAT-3' (Pribnow box), positioned at -10 with respect to the transcription initiation site -Mutations in any region diminish transcription, often severely