Molecular Biology: Translation and mRNA Codons

• These study notes cover key concepts in molecular biology.

Translation in Prokaryotes

• Removing the Shine-Dalgarno sequence from mRNA prevents the small ribosomal subunit from binding, thus affecting prokaryotic translation.
• The initiator tRNA is unable to bind to the A site without the Shine-Dalgarno sequence.

Transcription and Translation in Eukaryotes vs. Prokaryotes

• Bacteria can transcribe and translate the same mRNA simultaneously, while eukaryotes cannot.
• In eukaryotes, mRNAs are transcribed and processed in the nucleus.
• Translation happens in the cytoplasm.

mRNA Codons

• A codon is a triplet of three nucleotides on mRNA that codes for an amino acid.

Role of Release Factors in Translation

• Release factors recognize the stop codon and help dissociate the ribosome during translation.

Requirements for Translation

• mRNA, tRNA, and rRNA are essential for translation.
• RNA polymerase is not directly required for translation.

Function of Elongation Factors

• Elongation factors like EF-G shift the tRNAs in the A and P sites to the P and E sites during translation.

Blue/White Screening

• Blue/white screening indicates if colonies have a plasmid with additional DNA inserted.
• Blue colonies have a functional beta-galactosidase gene.
• White colonies do not have a functional beta-galactosidase gene due to the inserted DNA.

Expression Vectors

• Expression vectors require a promoter region before the multiple cloning site.
• It helps for transcription of the cloned sequence to occur in the host cell.

Bacteriophage Lambda and Cosmid Vectors

• Bacteriophage lambda and cosmid vectors can hold larger DNA fragments than plasmids.

Chain Termination Sequencing vs PCR

• Chain termination sequencing uses a single primer and a combination of dNTPs and ddNTPs, unlike PCR.

Chain Termination Sequencing and Dideoxynucleotides

• In chain termination sequencing, when DNA polymerase adds a dideoxynucleotide to the 3' end of the nucleic acid strand, chain elongation stops.

DNA Libraries

• If a DNA library is screened using a labeled DNA probe and the probe binds to DNA from two colonies, it indicates the colonies have sequence that hybridizes with the DNA probe.

Ion Torrent Sequencing

• Ion Torrent sequencing measures the change in pH caused by the release of a proton (H+) when a nucleotide is added.

Next Generation Sequencing Techniques

• Next-generation sequencing techniques like Illumina or Ion Torrent rapidly generate large amounts of sequence data by performing multiple sequencing reactions simultaneously.

dGTP vs. ddGTP

• dGTP has a 3'OH group, while ddGTP lacks it.

Cluster Formation in Next Generation Sequencing

• In next-generation sequencing, clusters are created to amplify the signal (fluorophore or pH) for better detection during sequencing.

Chain Termination vs. Illumina Sequencing

• Illumina sequencing uses reversible terminator nucleotides, and the sequence is collected as the reaction proceeds.

DNA Libraries

• Promoter sites for RNA polymerase are not found in the adapters added to DNA fragments when making DNA libraries for next-generation sequencing.

Importance of Separating DNA Clusters

• Separating DNA clusters allows only one sequence to be read at a time, preventing overlapping signals.

Finding Missing Sequences Between Contigs

• Missing sequences (gaps) between contigs can be found by screening another library (one that used a different cloning vector) with probe sequence from the ends of contigs

Exon Trapping Vectors

• When an unknown DNA piece is cloned into an exon trapping vector and a new sequence is observed between exons 1 and 2, it indicates an exon in the unknown DNA.

Sequencing Techniques Detecting Conductance Changes

• Third-generation sequencing with nanopore detectors detects changes in conductance as nucleic acids move through a membrane opening, converting these changes into sequence data.

Expressed Sequence Tags (ESTs)

• Expressed sequence tags (ESTs) are derived from transcribed regions of DNA.

Contigs

• Contigs are assembled by finding overlapping continuous sections of sequence.

Sequence Tagged Sites (STSs)

• Sequence-tagged sites (STSs) are unique sequences mapped to specific genomic locations.

DNA Replication Timing

• Cells copy their DNA only when preparing to divide.

Telomerase Function

• Telomerase maintains the ends of linear chromosomes in eukaryotes.

Location of STSs on Chromosomes

• When two STSs are often found together on the same fragment after chromosome fragmentation and sequencing, this indicates that they are located near each other on the same chromosome.

Replication Forks in a Replication Bubble

• Two replication forks are found in a replication bubble, with a replisome at each fork.

Semi-Conservative Replication

• All DNA replication results in two copies of double-stranded DNA, with each copy having a parent strand and a newly synthesized strand.
• This is known as semi-conservative replication.

Prokaryotic Promotors

• The -10 and -35 positions in the promoter are important for transcription in prokaryotes.
• They are AT-rich sites that interact with sigma protein to open DNA and position RNA polymerase.

Correcting Mismatches in Newly Replicated DNA

• The cell can identify the strand needing correction by methylation. The new strand is not yet methylated.

Leading Strand Synthesis

• Synthesis of the leading strand occurs continuously in the 5' to 3' direction.

Gyrase Function

• In prokaryotic replication, gyrase removes DNA supercoiling ahead of the replication forks.

Okazaki Fragments

• Okazaki fragments are joined to form a continuous strand.
• DNA polymerase removes the primer, fills in, and then ligase seals the nick.

RNA Polymerase Binding

• RNA polymerase binds to the promoter on the DNA to initiate transcription.

Information Transfer During Transcription

• During transcription, the information in DNA is transferred into RNA sequence.

Transcription

• When transcribing a DNA sequence into RNA, remember that Thymine (T) is replaced by Uracil (U).
• e.g. DNA: 5' ATGTGTGACGACGAT-3'; RNA: 5'-AUGUGUGACGACGAU-3'

Hairpin Loops and Termination

• The hairpin loop destabilizes the RNA-RNA polymerase interaction.
• The weak A=U bonds are easily broken to terminate transcription.

Untranslated Regions (UTRs)

• mRNAs have UTRs (untranslated regions).

RNA Processing

• Extra nucleotides are commonly included during intron splicing to generate more sequence diversity is NOT correct.
• Prokaryotic mRNA is polycistronic

Transcription Location

• Occurs in the cytosol in prokaryotes but in the nucleus in eukaryotes.

Inducers

• The activator binds to its sequence upstream of the promoter, assists the polymerase with binding, and allows for transcription.

Processing of tRNA

• Trimming removes nucleotides from the 5'- or 3'- ends of the transcript.

Prokaryotic vs. Eukaryotic

• In Eukaryotic transcription TFIID and TFIIB bind and help with positioning RNA polymerase 2 not -10 and -35 sequences like in prokaryotic transcription.

Eukaryotic mRNA Processes

• Phosphorylation of exon sequence is NOT performed on eukaryotic mRNAs.

snoRNA

• snoRNA acts as a guide for enzymes that will modify bases in the rRNA when base-paired with region of RNA.

Spliceosomes

• recognized by the snurps in the spliceosome base-pair with complementary sequence of the splice sites.

Operators

• Operator is the binding site of the repressor, which when bound prevents polymerase binding or moving along the template.

Antibiotic Resistance Markers

• What is the purpose of including an antibiotic resistance marker in a cloning vector?
• Allows you to keep all the cells with the vector while cells without the vector will not survive when grown in the presence of the antibiotic

Multi cloning Site

• Feature that allows you to clone your DNA into the vector.

cDNA Library

• The cDNA library is made from only expressed mRNA sequences.

Shuttle Vectors

• Have sequences that allow the vector to copy and be selected for in 2 different organisms.

Dideoxynucleotides

• ddATP is missing a 3'OH group

Contigs

• Contigs are assembled by finding overlapping, continuous sections of sequence.

Expressed Sequence Tags

• They come from transcribed regions of DNA

New DNA strands

• Are unable to tell if its new, detected by methylation. The new strand won't be methylated yet

Reverse Strand

• The New RNA sequence needs to be written in the 5' direction, remembering that Thymine (T) is replaced by Uracil (U).
  • e.g. 5'-TATTGCGTAGCA-3'; 5'- UAUUGCGUAGCA -3'

Topoisomerases during replication

• Enzymes that ligate the insert DNA with the vector DNA

Antibiotic resistance genes

• To select for cells that have taken up the vector

Real Time PCR

• Lets us calculate how much of an expressed sequence was present in a cell.

Vectors

• Unique region for inserting DNA into vector.

TaqMan Probe

• TaqProbe focuses on one sequence at a time instead of multiple sequences in the same sample

Transcription-PCR

• The gene/sequence of interest is expressed in sample A but not in sample B when it contains a product in sample A but not sample B

Site Directed Mutagenesis

• Intentionally introduces a mismatch in a primer to create the mutation.

Nonsense Mediated Decay

• Degrades mRNA's with premature stop codon so truncated proteins are not made