Cell and Molecular Biology: Transcription + Translation- Chapter 11.3

siRNA and proposed function

siRNA and proposed function

1. Small interfering RNAs that come from viruses, transposons, and protect the genome from those things by cleaving their RNAs

   1. Recognizing by base pairing, then drawing in enzymes [21-23 bp]

2. Double stranded

3. Protects genome from viruses and transpoosons (same ones they came from)

4. Cleaves RNA

miRNA and proposed function

1. MicroRNAS [21-24 bp]; double-stranded; they are coded in the genome in introns and intergenic “junk” genes/DNA

   1. This means that they are transcribed just like RNAs are

They give normal regulation of gene expression through suppression; either cleaving mRNA or blocking translation

piRNA and proposed function

1. Piwi-interacting RNAs; they are found in the genome and protect the genome from transposons in germ cells

   1. Not double stranded; unlike the other two

2. They are small; short RNAs; they are all inhibitory

3. Scientists use these in experimental manipulations, adding them to cells to block expression of certain RNAs, so we can understand what the RNAs do

What is CRISPR?

Short DNA segments that are identical to sequences from bacteriophage DNA

How is CRISPR used by bacteria?

Serve as a memory of past viral attacks, providing a defense by allowing the bacteriophage to be recognized and its DNA to be cleaved

How is CRISPR used by molecular biologists?

1. Biologists are able to use it to edit/modify the genome by making mutants

2. Engineered to work in eukaryotes (Cas9, sgRNA)

3. Creates double-strand breaks

4. Repair creates mutants of specific genes

5. Gene editing: insert new DNA sequences into specific sites

What is the function of other non-coding RNA?

Some people think other non-coding RNA is like simple background noise; meanwhile, others say it serves like yet-unknown regulator functions.

What is a codon?

The nucleotide triplets code for amino acids; 64 codons; 20 amino acids

3 Basic properties of the genetic code

1. The genetic code is triplet

   1. 3-bases

2. Non-overlapping

   1. Each nucleotide is only part of 1 codon

3. Degenerate/redundant

   1. More than 1 codon for a particular amino acid

4. Start of transcription is the methionine code (AUG); know 1 of the stops

Discuss the types of mutations

1. Synonymous mutations

   1. Changes in the nucleotide sequence that doesn’t affect the amino acid sequence, while nonsynonymous does affect the amino acid sequence

2. Mutation

   1. Change in the code, and possible mutations are single-base substitutions

      1. Sometimes synonymous, sometimes it is not, or sometimes nonsense (doesn’t code for amino acids anymore, now is stop codon and can destroy protein’s functionality); nonsynonymous mutation

   2. Or insertion (addition)/deletion of 1 or 2 bases

Causing frameshift mutations which shift the whole triple reading frame, changing all the following amino acids

2D Structure of tRNA

Upside down cross with knobs on the 3 shorter ends

3D Structure of tRNA

Helical upside-down L-shaped molecule

What is the anticodon?

The anticodon: 3 bases that interact/recognize in a complementary way with the mRNA codon

Where can the anticodon be found?

Found on the short center bottom cross arm (bottom of L)

Where does the amino acid bind?

The amino acid binds on the long, center, open cross arm

What is the importance of the unusual nucleotide bases?

1. Unusual bases are chemically modified bases found in closed cross loops and disrupt H-bond formation; serve as potential recognition sites for proteins

   1. (possibly making it so that amino acids can bind to the appropriate tRNA)

What is the importance of the invariant bases?

1. Found in every tRNA and generate the L-shaped tertiary structure

2. Aminoacyl tRNA synthases bring the amino acids to the right tRNA, recognizing the codon on the tRNA

Wobble hypothesis

The idea that it is the first 2 nucleotides for a codon are strict for that amino acid; however, the third can often be charged to code fort eh same thing and for the same tRNA

How does the wobble hypothesis work?

1. An anticodon U can bind an mRNA with A or G

   1. An anticodon G can pair with mRNA U or C, and I can pair with U, C, or A (all of this only in the 3rd position)

Why is the wobble hypothesis important?

1. This allows multiple different codons to work for the same trNA which reduces the number of tRNAs that are required. There is a wobble/variation in the way that the 3rd position of the codon interacts with the anticodon (so the same tRNA can recognize 2 codons with different 3rd positions)

1. *** KNOW 1 EXAMPLE OF A WOBBLE***

Describe the prokaryotic processes including any factors involved and explain where energy is required to drive the reactions: Initiation

1. Involves mRNA interacting with the small ribosomal subunit (disassembled) (the start codon: AUG/methionine)

2. How the mRNA is lined up with the small subunit), and various initiation factors (IFs) are involved; this includes IF3 which keeps the large subunit away and helps tRNA enter

3. IF1 stabilizes the mRNA and prevents tRNA from attaching incorrectly; IF2-Gtp brings in the tRNA, then the large subunit joins and GTP to GDP release IF2 (energy is released)(IF1 and IF3 also go away)

4. Steps

   1. IF3 prevents large subunit form joining, helps tRNA to enter

   2. IF1 stabilizes mRNA, and prevents tRNA from attaching to incorrect site

   3. IF2-GTP brings in tRNA

   4. Large subunit joints

   5. GTP to GDP releases IF2

Describe the prokaryotic processes including any factors involved and explain where energy is required to drive the reactions: Elongation

1. Involves new mRNA coming into the A (aminoacyl) site with the peptide being in the P (peptidyl) site,

2. Leaving via the E (exit) site, and EF-Tu-GTP (elongation factor) brings the new amino acid in (converting to GDP using energy; GTP to GDP)

3. Then, the short amino acid on the P site transfers to the A site to the peptide bond with the new amino acid (the original amino acid has a free amino (NH3) end, and the carboxyl end is what attaches to the new amino acid

4. We read it 5’ to 3’ and the directionality is amino to carboxyl, and the enzyme that makes the peptide bond is an RNA in the large subunit (ribozyme); ribosomal RNA (rRNA is the enzyme)

5. Once they are attached, they shift using EF-G_GTP (using energy)

6. The orientation of the large and small rachetes (rotates a little)

7. EF-G holding it in position so everything can fully move over and stabilize

Steps:

1. aa-tRNA entry

2. Peptide bond formation

3. Translocation

4. Release of deacylated tRNA

Describe the prokaryotic processes including any factors involved and explain where energy is required to drive the reactions: Termination

Involves a release factor (RF) which comes in when there is a stop codon (which codes for nothing), getting into the empty A site and triggering the release of the protein

Basic structural features of a ribosomes

1. Ribosomes are programmable machines; this means the info in their mRNA determines which tRNAs the ribosome accepts

   1. This takes palace as a repetitive cycle of mechanical changes driven by GTP hydrolysis energy release

2. Their component RNAs play a major role in selecting tRNAs, ensuring accurate translation, binding protein factors, and polymerizing amino acids

3. There’s part of the A, P, and E sites on both subunits

4. mRNA lines up on the decoding area of the small subunit, whereas the enzyme sites is on the large subunit, and when the 2 subunits are combined, the mRNA and tRNA enter a hole between them

Functions of a ribosome during translation

Multiple ribosomes can translate on the same long chain at the same time, each at different steps of translation and therefore, having different lengths of proteins

1. In prokaryotes, transcription and translation occur at the same time since there is no nucleus

What is nonsense-mediated decay?

The process by which mRNAs with mutations are identified and broken down, such that cells detect mRNAs which premature termination codons and selectively destroy mRNAs after only one translation