Genetics Test 4

Why Introns?

"Exon shuffling hypothesis: exon/intron organization may facilitate evolution of new genes... In some cases, exons of a gene code for a "functional domain"- a peptide region that assumes a useful conformation...e.g. binding a molecule -globin: heme containing region Do introns allow ability to "mix/assemble" different functional cassettes into new combinations?

nucleosomes

'beads on a string' in different stages of condensation

Output Assembly

1. Raw data is series of images 2. Each well's data is extracted, quantized, and normalized. 3. Read data converted into "flow grams"

Transcription in Eukaryotes

3 RNA polymerases responsible for copying DNA template --Pol I: Transcribes large ribosomal RNAs (nucleolus) --Pol II: Transcribes mRNA --Pol III: Transcribes most small RNAs, tRNAs and RNAs involved in processing primary RNA transcripts mRNA modificaton and processing primary transcript is modified in several ways before release into cytoplasm for translation --5' capping --3' polyadenylation --removal of intervening sequences (introns

The Polymerase Chain Reaction

Allows for amplification (essentially cloning) of small amounts of DNA from biological samples: provide specific primers flanking region of interest denature and anneal primers synthesize new DNA in vitro.... Denature and repeat annealing and synthesis 1>2>4>8>16 Procedure can be automated using DNA polymerases from thermophilic organisms...

Summary of Complementation Testing

Amenable to wide variety of organisms Test for whether mutant genes lie in the same functional unit --If 'trans' configuration leads to function: genes lie in different functional units- different 'cistrons', distinct genes --If 'trans' configuration leads to mutant phenotype: gene lie in same functional unit- they are alleles

What distinguishes a cDNA library from a Genomic DNA Library? A) A cDNA library should contain all protein coding genes B) A cDNA library will contain DNA sequences from those genes that are expressed in the cells used for library preparation- not necessarily all genes in the genome C) A genomic library contains only non protein coding sequences D) All the above E) none of the above

B) A cDNA library will contain DNA sequences from those genes that are expressed in the cells used for library preparation- not necessarily all genes in the genome

Correct labeled amino acid leads to ribosome assembly

Binding of correct charged tRNA promotes ribosome assembly and traps radioactivity on filter If the incorrect anticodon was used in the assembly reaction, no radioactivity would be trapped on filter

RNA editing

Changes introduced into specific nucleotide sequences within a RNA molecule after it has been generated by RNA polymerase. First described following evidence for the insertion of uridine (U) residues into a mitochondrial mRNA of Trypanosoma brucei. Genes transcribed from the main mitochondrial genome (maxicircle) were edited using guide RNAs derived from minicircle templates...

Deletion Mapping of rII mutants

Collected many individual point mutations crosses testing every mutant against each other too time consuming first mapped 7 large deletion mutants against each other and against 47 smaller deletions

construction of cDNA libraries

Convert mRNA sequence into double-stranded DNA DNA 1. Transcription PremRNA 2. RNA processing: introns removed mRNA 3. Translation Polypeptide --provides a "snapshot" of the genes expressed in a particular tissue --isolate mRNA from tissue of interest --convert to d.s. DNA in vitro with reverse transcriptase and Pol I -- prepare ends for cloning (remove hairpins, add ligate on restriction sites) -- clone into vector and propagate in microorganism

The Code is Universal (Almost)

Cytoplasmic organelles (mitochondria and chloroplasts) contain their own genomes These molecules are usually present as a closed circular DNA molecule These genomes encode their own rRNA and tRNAs and have their own translation apparatus

Proteins and Phenotype: enzymes are gene products

DNA protein --Archibald Garrod (early 1900's): defects in metabolism are caused by recessive genes --"inborn errors of metabolism" --Beadle and Tatum: Neurospora --Their studies in mid-1940's demonstrated that gene mutations led to defects in specific biochemical pathways --"one gene- one enzyme" hypothesis Won Nobel Prize, 1958; shared it with Lederberg But using genetics to explore enzyme pathways wasn't done until the 1940s... And it took close to 100 years before Garrod's mutation was characterized at the molecular level

Using 'replica plating and nucleic acid hybridization to isolate specific clones from recombinant libraries

Each clone occupies a specific place on the plate (can screen ~50,000 plaques per plate) 1. replica plate plaques or colonies onto filter 2. denature DNA on filter 3. incubate with radiolabeled probe...wash off unbound DNA 4. detect cells that contain correct gene by autoradiography 5. grow up appropriate clone

True or false. Since E. coli has a single copy genome, it is impossible to isolate a mutation in a gene encoding a protein required for a critical function like DNA replication, since such mutations would be expected to be lethal.

False

Chromatin Structure

Histones are highly conserved proteins that are intimately associated with the DNA in chromatin --small in size --carry a large number of basic residues --;complexed into a particle termed a nucleosome

Summary of Genomic library construction

How do we clone a gene? 1. Extract DNA from organism 2. Extract vector from bacteria 3. Open up vector 4. Cut DNA into fragments 5. Make recombinant DNA 6. Introduce into bacteria to clone

Screening using related sequences

If a tissue is enriched in a particular mRNA, the mRNA could be used as a probe --e.g. globin gene sequences Sometimes it is easier to isolate a sequence from one organism than another, and the related gene can be used to fish out the same gene from other genomes --e.g. actin gene sequences:

Rho factor: factor mediated termination

In an ATP-mediated reaction, a rho protein complex binds to the mRNA and unwinds RNA from the DNA template recognition sites may not have hairpins or U tracts; tend to be C-rich

How does the configuration of chromatin affect gene expression?

It would seem to be difficult to transcribe DNA that was complexed in the nucleosome core; RNA polymerase appears to displace histone octamers during transcription It is possible to distinguish "active" from "inactive" chromatin by susceptibility to enzyme digestion --In liver chromatin, beta globin gene is not expressed- beta globin gene DNA LESS susceptible to DNAse digestion --In reticulocyte chromatin, beta globin gene expressed- MORE susceptible to DNAse digestion.

DNA (Gene) Protein Experiments of Seymour Benzer

Link between classical concept of the gene and molecular concept of the gene Three Basic Question addressed in experiment --What is the basic element of structure? --What is the basic element of change? --What is the basic element of function?

produced during lagging strand synthesis

Okazaki fragment

Different Vectors are Engineered for Different Purposes

Plasmids: up to15 kb Phage: up to 25 kb Cosmids: 30-45 kb BAC: 100-500 kb YAC: 250-2000 kb

region of DNA associated with prokaryotic promoters

Pribnow Box

Frameshift mutations

Proflavin dyes: molecules have the dimensions of a purine-pyrimidine base pair Distortion in helix will cause errors during DNA replication; leads to addition or loss of a base pair

DNA RNA: Transcription

Prokaryotes: E. coli model system RNA polymerase --Initiation: promoter binding and transcriptional control --Elongation: transcription of polycistronic mRNAs (mRNA encoding several proteins) --Termination: release of RNA polymerase and mRNA Transcription of gene is asymmetric...only one strand is copied But on any chromosome, genes are transcribed from both strands

Protein Structure

Proteins are polymers built from amino acid subunits... 20 different amino acid side chains are found in proteins Proteins are assembled with the formation of a peptide bond between the carboxyl group of one amino acid and the amino group of another... - Assembly occurs on the ribosome

Beadle and Tatum (~1940s):

Selection scheme for biochemical mutants in the same biochemical pathway --mutagenize strain and mate --dissect out haploid spores --grow on complete media --test for ability to grow on a minimal media -- test on a more defined media to isolate mutants in common pathway

Linear sequence of amino acids in chain... primary structure

Side Chains (R groups) project from the backbone Protein chains, like DNA, have a polarity (NH2 COOH)... a repeating peptide backbone (C-C-N) Parts of the backbone are held rigid due to limitations on bond rotation Rotation around the alpha carbon allows proteins to adopt a distinct three dimensional shape limitations on rotation dictated by primary sequence influence folding

Array technologies can also be used to monitor for polymorphisms in the DNA

Single Nucleotide Polymorphisms (SNPs) can be detected by this technology...allele specific oligonucleotides (ASO)

Features of the Code

The code is "degenerate": i.e. different codons can specify the same amino acid A substitution at the third codon position often encodes the same amino acid amino acids related in their properties have similarities in their codon structure; e.g.: --acidic residues are GAN (N = G, C, U or A) --all codons with a U in the second position are hydrophobic

"Cracking the Code" Translate the synthetic mRNA in an in vitro protein synthesizing system.

The frequency of the amino acids made should reflect the codon frequency: phe (UUU) = 42% leu (UUG) = 14% val (GUU & GUG) = 19% cys (UGU) = 14% trp (UGG) = 5% gly (GGG & GGU) = 6%

Genetic Selection can also be used in screening

The gene must be introduced into a vector so that the information encoded within it can be expressed If the gene can complement a mutation in the cell into which it is being transformed, then that is a powerful means of isolating the gene of interest- only the clone of interest would survive If the gene can produce a protein in the cell, then production of that protein product can identify clone

Codon "Wobble"

The number of distinct tRNA anticodons can be less than the number of possible codons Because of relaxed base pairing interactions occurring at the third position of the codon and the 1st position in tRNA anticodon, some tRNAs can recognize more than one codon

Translation in Prokaryotic Systems

Three major components necessary to translate information in mRNA molecule - mRNA - ribosome - charged tRNAs

True or False: The large ribosomal subunit acts as a 'ribozyme'- the peptidyl transferase activity that forms the peptide bond during translation is associated with the RNA component of the large subunit, not the protein component

True

True or false. Some genes are transcribed, but not translated.

True

Information on protein sequence can lead to the synthesis of short oligonucleotide probes

Use nucleic acid hybridization to identify complementary gene sequence

Cosmids

Vectors that are hybrids between plasmids and lambda phage can replicate in a cell like a plasmid or be packaged like a virus they can carry larger DNA inserts than plasmids

The alphabet of 20 different amino acids contains subunits that are

acidic basic contain atoms that can form H-bonds contain hydrophobic residues contain reactive groups

Primary transcript

contains both introns and exons...introns must be removed

DNA Sequencing: the Chain Termination Method

dideoxynucleotides used to stop synthesis at a specific base... --4 different DNA synthesis reactions are run --begin synthesis from specific priming point --add components for DNA synthesis + a specific ddNTP for each of the four reactions...e.g. ddATP reaction is shown here... --Fragments of a discrete size accumulate in each reaction... --Run the 4 different reactions on electrophoretic gel...smallest fragments (closest to primer) migrate farthest...

A specific retrovirus has a single-stranded RNA genome consisting of 18% guanine. The percentage of uracil in this organism's RNA would be: a) 18%; b) 25%; c) 32%; d) 36%; e) cannot be determined from the information given.

e) cannot be determined from the information given.

hypermutable site

hot spot

used to localize genes to specific bands in eukaryotic chromosomes

in situ hybridization

uses thermostable enzyme to amplify DNA sequences

polymerase chain reaction

causes mutations by intercalating into DNA

proflavin dyes

protein complex that protects chromosome integrity, preventing non-homologous end joining

shelterin

SNRPS:

small nuclear ribonucleoprotein particles

Timeline

1902: identified as Mendelian trait 1908: "inborn error of metabolism 1958: homogentisate 1,2 oxidase 1992: mapped genetically (linkage) 1995: cloned in fungus 1996: human gene cloned and used as probe, mutations in DNA identified by PCR

Massively Parallel Sequencing Emulsion PCR

A single DNA fragment is amplified in a 'microreactor' --Water-in-oil emulsion generates millions of micelles. --Each micelle contains all reagents/templates for a PCR reaction. --~10 Million individual PCR reactions in a single tube.

Polymerase chain reaction: A) Requires DNA to be denatured after every round of the cycle, so specific primers can be annealed B) Requires DNA polymerase to be added after every denaturation cycle, since its destroyed by heat denaturation C) Can produce almost a million amplified DNA products after 30 cycles D) All E) None

A) Requires DNA to be denatured after every round of the cycle, so specific primers can be annealed

How does tRNA become charged with an amino acid?

Aminoacyl tRNA synthetases charge tRNAs in a two-step reaction. Reaction must be specific...different tRNA synthetase enzymes charge the appropriate tRNA with the appropriate amino acid

Next Generation Sequencing Technologies

Are able to produce sequence data in much larger volumes and at much lower costs Will allow individual sequence information to be collected much more easily --Cost of human genome project: ~$2.7 billion --Goal of next generation sequencing: the $1000. genome --5/12 sequencing costs using next generation technologies: around $9,500/genome --~$6,500 - $1,500 today

Restriction fragment length polymorphisms (RFLPs)

Autoradiograph of Southern blot produced from previous genomic DNA restiction digest, probed with a muscle actin gene probe Homozygotes DNA polymorphism: 3 different "genotypes"

Illumina Sequencing at OMRF (OKC):

Average length of read: 200 bases >60 million paired end reads/lane Cost of run: ~$1500 (reagents, supplies, labor) 8 x 106 bp/ dollar >106 fold increase in sequencing capacity relative to conventional sequencing

454/Roche GS-FLX at OU ACGT:

Average length of read: 600-800 bases 800,000- 1,000,000 high quality reads/plate ~800 Mbp/10 hour run time Cost of run: ~$12,000 (reagents, supplies, labor) 67,000 bp/ dollar >1000 fold increase in sequencing capacity relative to conventional sequencing (96 well, 800 nt read, 2.5 hr run time)

Which of the following is required for the construction of cDNA library that would represent genes expressed in the frog pituitary gland? A) Frog genomic DNA isolated from pituitary cell nuclei B) Fragmentation of frog genomic DNA with restriction enzymes C) Reverse transcriptase D) All the above E) None

C) Reverse transcriptase

Which of the following are characteristics of prokaryotic translation? A) Translation occurs after the mRNA is released to the cytoplasm B) An initiation codon is located at the beginning (extreme 5' end) of the mRNA transcript C) The mRNA start codon is placed at the P site of the ribosome D) All E) none

C) The mRNA start codon is placed at the P site of the ribosome

7. E. coli DNA polymerase III: a) synthesizes a DNA chain in the 5' to 3' direction; b) is a multisubunit protein complex at the replication fork; c) has a 3' to 5' exonuclease activity; d) all of the above; e) none of the above

D) All the above

What features of plasmid cloning vectors are important for their use in cloning recombinant DNA molecules? a) they contain a dominant selectable marker; b) they contain an origin of replication; c) they contain a restriction site in a nonessential region of the plasmid genome; d) all of the above; e) none of the above.

D) All the above

Northern blot hybridizations

Detection of gene expression in different tissues Hybridize with gene-specific probe Separate different mRNAs on gel, and transfer to filter

Cracking the Code: Khorana

Developed a way to chemically synthesize short stretches of ribonucleotides in an ordered array: e.g. --(UG)n = UGUGUGUGUGUG... --could give rise to two different alternating codons UGU GUG UGU... --led to synthesis of alternating polypeptide cys-val-cys-val...

Suppose the polynucleotide phosphorylase was used to produce a synthetic mRNA using a ratio of 3:1 G to U: The fraction of the nucleotide chain that would encode the codon 'UUG' would be approximately A) 42% B) 33% C) 25% D) 14% E) 5%

E) 5%

Example of protein structure: lysozyme

Enzyme binds and cleaves a carbohydrate component of bacterial cell wall, causing cell lysis enzyme activity depends on the appropriate three dimensional folding of enzyme: formation of binding site and catalytic site Lysozyme contains a number of domains of discrete secondary structure

Central Dogma DNA --> Protein

Evidence for an RNA intermediate in information transfer (Ch 8): --prokaryotes: on phage infection, short-lived RNA molecules could be detected. Similar in base composition to phage genome, not the host genetic material -- eukaryotes: 'pulse-chase' experiments indicated an RNA molecule was transported from nucleus to cytoplasm...

Prokaryotic Translation: production of a protein from mRNA

Initiation requires the assembly of the mRNA on the ribosome at the appropriate starting point for translation -- A specialized charged tRNA associates with the small subunit -- The large subunit is recruited to finish the complex; first tRNA is present in "P" site of ribosome

RNA-Seq Transcriptome Analysis

Isolate mRNA from tissue of interest Convert mRNA population to cDNA Use next generation sequencing to profile variety and number of individual transcripts

Several types of stable domains of secondary structure, based on H-bonding within chain, exist

Local regions of folding, based on hydrogen bonding interactions, introduce domains of stability as the protein assumes a 3 dimensional shape Strong hydrogen bond: Linear (all atoms in line.) Weak hydrogen bond: Bond forms at angle As regions of secondary structure coalesce, individual polypeptide protein chains assume their final three dimensional conformation, tertiary structure If two (or more) individual proteins come together, their mutual interaction can further change 3 dimensional conformation, leading to quaternary structure

"Cracking the Code"

Make a synthetic mRNA Produce a protein synthesizing reaction in a test tube (in vitro) from components isolated from bacteria (ribosomes, charged tRNAs, appropriate salts, buffers) --add synthetic "mRNA" --test to see if radioactive amino acid is incorporated into proteins With PNP, composition of RNA is random and synthesis will depend on amount and type of nucleotides added to reaction --e.g. all U's will make UUUUUUUUUU... --all G's will make GGGGGGGGG Translation of synthetic RNA could be accomplished in a test tube using extracts made from E. coli cells: e.g.: --Make E. coli extracts from cells grown in the presence of one (out of 20) "hot" (radiolabeled) amino acids ---these 20 different lysates will contain ribosomes and charged tRNAs, one of which will be radioactive --add synthetic "mRNA" --test to see which radioactive amino acid is incorporated into proteins - UUUUU....= Phe-Phe-Phe... - GGGGG....= Gly-Gly-Gly...etc - Make synthetic RNAs using combinations of nucleotides: e.g. 3/4 U, 1/4 G: GUUUUGUUUGUU... (random polymer) 8 possible codons: UUU,UUG, UGU,GUU, UGG, GGU, GUG, GGG The frequency of the 8 codons will depend on the ratios of U and G in the synthetic mRNA: UUU = 3/4 X 3/4 X 3/4 = 27/64 = 42% UUG = 3/4 X 3/4 X 1/4 = 9/64 = 14% UGU = 3/4 X 1/4 X 3/4 = 9/64 = 14% GUU = 1/4 X 3/4 X 3/4 = 9/64 = 14% UGG = 3/4 X 1/4 X 1/4 = 3/64 = 5% GGU = 1/4 X 1/4 X 3/4 = 3/64 = 5% GUG = 1/4 X 3/4 X 1/4 = 3/64 = 5% GGG = 1/4 X 1/4 X 1/4 = 1/64 = 1%

Hybridization Technologies: Examination of differences in gene structure and expression

Review of Southern Blotting Review of Northern Blotting Microarray Analysis

Cracking the Code: Nirenberg

Ribosome binding assay: under in vitro conditions, the two subunits of a ribosome and the appropriate tRNA can assemble on a short RNA consisting of just a single codon triplet. --Synthesized all triplet combinations. --Tested to see which charged tRNA (out of the twenty possible) could lead to the assembly of a ribosome complex

Hybridization to microarrays

Single-stranded DNA molecules can be fixed to a solid matrix at defined positions on a grid; if oligo-nucleotides are used, tens of thousands of genes can be represented and sampled together The gene expression patterns in the two samples can therefore be compared -- cDNAs made from two different RNA populations, labeled with different colors, can be hybridized simultaneously to the entire set of "genes" represented on the array -- Similar levels of gene expression in normal and tumor cells (gene expressed in higher levels in both norman and tumor cells) Array experiments can give information on networks of genes that may be expressed togethe -- transcriptome analysis

What is the basic unit of recombination?

Size of genome as detected by electron microscopy: 2 x 10 5 bp size of T4 linkage group as determined from intergenic mapping studies: 1500 map units closest mutations mapped by Benzer: 0.02 map units nucleotide is the basic unit of recombination (i.e., structure- recombination can occur within genes) mutation can occur at the nucleotide level; individual mutations can occur at the resolution of a single base pair the distribution of mutations need not be random within the nucleotide sequence; some nucleotides may be more susceptible to mutation than others (hot spots) when induced mutations were examined, there were also hot spots, although often different than the spontaneous ones

THL

The amino acid sequence (i.e. DNA-encoded primary structure) can dictate the correct folding of a protein molecule in 3D space

The sigma factor of E. coli is required for which function? a) binding to a promoter; b) polymerizing an RNA chain; c) releasing the RNA from the DNA template; d) all of the above; e) none of the above.

a) Binding to a promoter

Elongation

assembly of a polypeptide on the ribosome -- charged tRNA enters the A site via pairing to codon -- peptidyl transferase activity on large subunit forms peptide bond, transferring growing peptide to A site -- translocation of mRNA relative to ribosome moves nascent polypeptide to P site

Promoter

binding site on DNA for RNA polymerase the sequence at the promoter can regulate efficiency of initiation different sigma factors may associate with RNA polymerase, which target specific promoters

Specific Transcription factors

can influence the formation of a productive transcription complex... --STFs may bind at some distance from the promoter...enhancer sequences

Southern Blotting to detect genetic polymorphisms

cut DNA with restriction enzyme run DNA on gel to sort fragments by size denature DNA and transfer to filter hybridize with single stranded probe wash away unbound probe and expose to X-ray film...

Which of the following would be used for the construction of a cDNA library, but not a genomic library? a) restriction enzymes; b) DNA ligase; c) vector DNA; d) reverse transcriptase; e) transformation.

d) Reverse transcriptase

In bacteriophage lambda, the two strands of DNA can be separated from each other on the basis of their density, using denaturing CsCl gradients and ultracentrifugation. mRNA synthesized by lambda phage can be isolated from infected cells, and when allowed to anneal to the denatured DNA, it is observed that both DNA strands are capable of forming mRNA-DNA duplexes. This observation indicates: a) bacteriophage lambda contains one long polycistronic mRNA; b) the two strands of lambda DNA must be identical in sequence; c) all genes in lambda are transcribed off the same DNA strand; d) both strands of the lambda DNA can serve as a template strand for transcription; e) none of the above.

d) both strands of the lambda DNA can serve as a template strand for transcription;

rII mutant phenotype

difference in plaque morphology; produces large plaques on strain B E. coli also difference in host range; can not grow on E coli strain K12; can grow on E. coli strain B

Automated DNA sequencing:

each reaction can be specifically labeled with a fluorescently tagged molecule since each terminated fragment is tagged with a different color, all reactions can be conducted and run on the same lane a laser built into the gel apparatus can now illuminate the bands and a spectrophotometer can record the wavelength and intensity of the fluorescent signal as the DNA band passes the detector Blue fluorescent signal detected for C base Violet fluorescent signal detected from G base

Origin of Organelle Genomes and Non-Nuclear Inheritance

endosymbiont theory: organelles represent remnants of free-living organism that established symbiotic relationship with proto-eukaryotes (Lynn Margulus) inheritance of organelle genetic information is often uniparental, usually maternal Mechanism of inheritance will therefore be non-Mendelian, often only through egg cytoplasm (e.g. MERRF - tRNA lys mutation)

General (basal) Transcription Factors

interact with the promoter to facilitate binding of RNA polymerase II...e.g.

TBP: TATA binding protein (TFIID)

is capable of interacting with several factors that are required for the assembly of a productive transcription complex

Higher order folding

loops of DNA are believed to associate with a protein scaffold

Termination

mediated by a protein release factor -- stop codon (UAG, UGA, UAA enters "A" site) -- release factor interacts with stop codon -- polypeptide chain is released and ribosome dissociates

Evidence for a triplet code

numerical argument: to encode 20 amino acids, a code word of at least 3 is necessary (41= 4; 42 = 16; 43 = 64) experimental support: Intragenic suppressor mutations in T4 rII locus indicated a triplet code...experiments of Crick and Brenner (pp. 259-61/249-52)

Exon

part of the primary transcript that remains in the mature mRNA

the linkage between R groups to form a polymer

peptide bond

General Structure of tRNA molecules

small; approximately 75 nucleotides extensive intrastrand hydrogen bonding "cloverleaf" structure contains 3 characteristic loops; anticodon loop is responsible for pairing with mRNA 3' end of molecule associates with specific amino acid

Intragenic suppressor mutations

suppose one mutation added a base... incorrect frame leads to mutant suppose a second mutation removed a base... reading frame restored... if sites are close together, phenotype may be near normal the existence of separate sites could be shown by recombinational analysis with wildtype T4

telomere repeated consensus sequence of [TTGGGG]n is replicated by special enzyme

telomere

tRNA

the adaptor molecule for protein synthesis: mRNA --> protein tRNAs are the adaptor molecule that will link the genetic code to a specific amino acid

C. Yanofsky

the code is non-overlapping, and there is colinearity of mutations in genetic map with amino acid changes in mutant proteins Individual point mutations only change a single amino acid... evidence for non-overlapping code

relieves torsional stress during DNA replication

topoisomerase

Bacteria grow extremely rapidly relative to eukaryotic cells

transcription (mRNA synthesis) and translation (protein synthesis) are coupled Even prior to the conclusion of transcription, ribosomes will assemble on nascent (i.e. developing) mRNA, and begin to synthesize protein.

S. Ochoa

use of the enzyme polynucleotide phosphorylase (PNP) to make synthetic RNAs