Unit 2

Translation Elongation:

(cycle for every amino acid that gets added to chain until STOP codon is reached) 1. amino acid chain in P site 2. codon recognition by next tRNA which comes into A site (helper proteins which bring tRNA to ribosomal A site) 3. GTP hydrolysis to power the formation of new peptide bond between the existing amino acid chain on the tRNA in the P site with the new amino acid 4. GTP hydrolysis to move translocate ribosome down one codon and push tRNA with no polypeptide chain out of exit site

Explain the generation and components of ribosomes.

* composed of proteins and RNAs * have transcribed spacers in between regions preserved in rRNA * processing down to actual ribosome happens from nucleolus to cytoplasm * protein components made in cytoplasm and transported into nucleus to make ribosomes

Introns are 'junk' DNA that create a burden on the species. Give at least two reasons why this statement is incorrect?

Alternative Splicing Some introns have an effect on gene regulation

Discovery of Transposons:

-1st experimental proof that genes were physically positioned on chromosomes by studying crossing over and recombination in Zea Mays (corn). -Studying transient loss of function in kernel color genes in corn lead to the discovery of mobile DNA elements (Gene for purple corn kept moving around).

Transformer Protein

-U2AF in males causes a truncated, nonfunctional Transformer Protein -Sxl in females allows creates functional transformer protein; outcompetes splicing proteins -example of alternative acceptors

Repressor-Activator Competition in HIV

-have both ESE and ESS -A1 repressors block U2AF from binding -SC35 activator allows U2AF -allows for both types of transcripts to be produced -competition between repressors and activators -want both transcriptions to be produced in some ratio

For most eukaryotic mRNAs, the absence of a polyA tail will:

-probably prevent it from leaving the nucleus. -shorten the half-life of the RNA. -reduce the efficiency of its translation. NOT reduce the efficiency of its splicing.

Approximately 45% of the human genome is derived from transposable elements. How is survival possible with this high a percentage of transposable elements in the human genome?

1) Transposable elements inserted in introns are positively selected and do not harm gene expression. 2) The majority of elements are inactive due to the accumulation of mutations. Therefore, they can no longer jump into vital genes. 3)The active elements that are capable of increasing in copy number are rendered inactive by host regulatory mechanisms.

Eukaryotic Transcription Initiation and Elongation

1. A eukaryotic promoter includes a TATA box 2. Several transcription factors must bind to the DNA before RNA pol can do so. 3. Get RNA polymerase into dsDNA by creating torqued structure. 4. Additional transcription factors bind to the DNA along with RNA pol, forming the transcription initiation complex. 5. Kinase and other transcription factors come in and phosphorylate polymerase CTD tail to allow elongation to begin; activate RNA polymerase. 6. Transcription factors leave. 7. Addition of nucleotides through elongation.

Transposon Duplication mechanism:

1. Before S phase: One copy of transposon 2. S phase: DNA replication and transposition from behind replication fork to in front of the replication fork. 3. After S phase: one daughter molecule has 2 copies of transposon

5' capping:

1. CTD tail associates with capping enzymes to localize the capping enzymes to add the 5' cap while the transcript is coming out 2. Guanosine (3 phosphates) with 5' phosphate tail and 5' end of mRNA (with 3 phosphates) 3. connect using phosphate groups to form 5' to 5' triphosphate bridge 4. Methyl group is added to guanine to create modified guanine with methyl addition (7-methylguanosine)

RNA-sequencing (RNA-seq)

1. Isolate RNA from 2 different samples 2. RT-PCR to make cDNA library 3. chop up cDNA 4. sequencing and alignment of sequencing reads 5. see how many pieces of each sequence did you get that match each transcript

Describe the process of nuclear export.

1. NPL3 protein binds to ssRNA transcript as it is being synthesized and is phosphorylated 2. Once Poly (A) tail is added to RNA transcript, Glc7 phosphatase dephosphorylates NPL3 3. NXF1/NXT1 (export proteins) bind to NPL3 and take it with RNA transcript to nuclear pore complex and export it from nucleus 4. Dissassociation of proteins from RNA transcript

Translation Termination:

1. Ribosome reaches a stop codon on mRNA causing a release factor to bind to the stop codon (with the help of helper proteins) 2. Release factor promotes hydrolysis between the tRNA and the amino acid chain 3. GTP hydrolysis to disassemble ribosome 4. Ribosomal subunits and other components disassociate.

Translation Initiation:

1. Small ribosomal subunit binds to mRNA and finds Met site (start codon) and initiator with tRNA binds to that spot (only one that can bind with only small subunit present) 2. Initiator factors help binding of initiation tRNA to small ribosomal subunit 3. GTP is used to power the addition of the large ribosomal subunit which complete the initiation complex (initiator in tRNA starts in P site)

Mircoarray

1. Take cell and grow on culture 2. Isolate mRNA 3. Reverse transcription and labeling of cDNA using fluorescence 4. Hybridize cDNA probes to oligo sequences on microarray 5. Scan to compare ratio of fluorescence to find which transcript was present in which cells

Promoter Specificity Experiment Mechanism:

1. Take promotor sequence of a gene and put it in front of a reporter gene (GFP/luciferase) in DNA. 2. A reporter protein is made and the amount is measured. 3. Allows one to find where and how much of a specific gene is being expressed.

mRNA processing mechanism:

1. Transcription, 5' capping 2. Cleavage at poly(A) site by endonuclease 3. Polyadenylation by Poly(A) polymerase (PAP) + ATP 4. RNA splicing

Describe the process of mRNA splicing.

1. snRNPs are combination of snRNA and proteins which come together to form the spliceosome (U1 binds to pG on 5' exon and U2 binds to branch point) 2. First transesterification cleaves bond between 5' exon and the intron and glues intron end to itself at branch point 3. Second transesterification results in 2 spliced exons and the lariat intron

Wobble position

5' in anticodon

What is meant by "functional RNA"? What kinds of functional RNA are well-known and what do they do?

A functional RNA is an RNA that itself performs and actual function, not just as a template for protein synthesis (like mRNA). Ribosomal RNAs (rRNAs) are major components in ribosomes, transfer RNAs (tRNA) bring the amino acids to the ribosome for protein synthesis, snRNAs are components of the splicesome, etc.

How is exon shuffling beneficial?

In terms of evolution

Silent Mutation

Nucleotide substitution with no change in amino acid chain

Which RNA pol do you think binds to the most different promoters in the cell? Which RNA pol is the most active in the cell? Why?

RNA pol II binds to more different promoters because there are way more protein coding genes than there are any of the other RNA types. RNA pol I is the most active because ribsosomal RNAs are big and constantly being transcribed. There are a TON of ribosomes in a cell.

What is the difference between RNA polymerase and DNA polymerase?

RNA pol creates RNA molecules but DNA pol creates DNA molecules

Compare and contrast the advantages and disadvantages of RNA-seq and qPCR. Think about the information gained, workflow, and requirements.

RNA-seq can provide knowledge of novel alternative splicing transcripts and novel splice sites. RNA-seq gives information about transcript levels across the entire trancriptome rather than single genes. RNA-seq generally has a longer lead time, and produces a ton of data that has to be analyzed. If you are only interested in a few genes qPCR is much more time and cost effective. When done properly qPCR is much more accurate. Can be done without assembling an entire genome sequence.

Quantitative RT-PCR

RT= reverse transcriptase 1. RNA molecule (mRNA) extracted 2. reverse transcriptase used to make DNA copy of all RNA (cDNA synthesis) 3. Use PCR to amplify DNA with addition of fluorescent dye 4. Emission of the fluorescence increased by binding to DNA 5. See how much DNA is present after every cycle of PCR to see how DNA you started with 6. the more DNA, the brighter the fluorescence 7. the quicker you reach threshold, the more DNA was present initially

LINE Retrotransposon mechanism:

SYNTHESIZES RNA MOLECULE AND THEN INCORPORATES IT AND THEN REPLACES IT

LTR Retrotransposon Mechanism:

SYNTHESIZES dsDNA THEN INCORPORATES IT

Your boss asks you to focus on the splicing events of human gene X. Luckily, from previous work in your undergraduate lab, you already know the sequence of the entire gene. How can you experimentally determine where the introns and exons are in gene X?

Sequence the mRNA and compare the sequence to the genomic DNA sequence. Anything present in the mRNA is exon, anything absent is intron.

Retrotransposons move via an intermediate that is:

Single-stranded RNA

Some viruses shut down host cell gene expression by inhibiting all nuclear import. Why would gene expression shut down if nuclear import was inhibited?

Because Proteins that are important in gene expression processes in the nucleus are produced in the cytoplasm.

Transposase restriction mechanism of IS element restricts the transposon and the target DNA in a combination of which of the following?

Blunt end cut for transposon and sticky end cut for target DNA

What is so special about RNA that makes it such an attractive precursor to DNA and protein? What is it about DNA that makes it a better material than RNA for storage of genetic information?

DNA is much more stable than RNA, this means that information that needs to be stored long term is better off in DNA form. For mRNA, we want relatively high turnover so that gene expression/protein levels can constantly be adjusted.

You utilized a transposon system for a forward genetic screen, creating random disruptions throughout the genome and screening for phenotypes. You found a mutant with a really interesting, never before seen phenotype. How could you figure out what gene is affected?

If you know the sequence of the transposon that you inserted, which you should since you chose to use it in your experiment, you can design a primer to amplify out from your transposon and then sequence the product. Your product will contain part of the transposon sequence then DNA from the organism's genome where the transposon inserted. As long as the organism's genome is sequenced you should be able to identify a location. The more costly approach is to sequence the organism's entire genome and then look for the transposon you inserted, as long as it isn't naturally occurring in the organism. It is possible/likely that you will see multiple insertions but you can narrow it down from there using other methods. By doing PCR put from the transposon sequence itself to identify the DNA adjacent to it. You could also sequence the whole genome (but that is a little extreme.)

The TATA box is found in most eukaryotic promoters, but not all. What must be different about transcription for these genes?

They must have other specific sequences that are recognized by different transcription factors that can help initiate transcription.

Why do transposons preferentially integrate in transcriptionally active regions of DNA?

Transposons must have access to the DNA in order to insert into it. Transcriptionally active DNA is more frequently unpackaged and accessible to the transposon machinery.

A particular triplet of bases in the coding sequence of DNA is AAA. The anticodon on the tRNA that binds the mRNA codon is

UUU.

What genetic phenomenon explains the following paradox: There are 64 possible codons, but only 30-40 tRNA types.

Wobble

A promoter is:

a DNA sequence to which RNA polymerase binds.

GUS Staining:

a type of experiment to test promoter function * ethanol used to rid plant of natural pigment * turns blue in areas where gene is turned on. * more stable

Luciferase Reporter Experiment:

a type of experiment to test promoter function * lets you get clear dynamic responses * transitory, enzymatic process

Steps performed by the enzyme transposase during transposition of bacterial insertion sequences:

a. excision of the IS element from the donor DNA molecule. b. introduction of staggered cuts into the target DNA molecule. c. ligation of the IS element to the target DNA.

Frameshift causing immediate nonsense

addition of extra nucleotide resulting in a stop codon causing the stop of translation

Alternative splicing and polyadenylation

changing where the end is (which is the last exon)

ORF1 in Line retrotransposon:

codes for RNA binding protein

ORF2 in Line retrotransposon:

codes for Reverse transcriptase

Transposons in Research:

commonly used to create random mutations for genetic screens 1. Introduce transposon direct repeating sequences which allow transposon to move 2. Introduce phenotypic marker 3. Separately introduce transposon

Intron Retention

considered intron for one product but not for others

Mutually exclusive exons

either one of two exons (never both)

Cassete Exons

exon in the middle which can be skipped

sigma factor in prokaryotes

helps recruit and bind RNA polymerase to promoter (helps process get started then leaves)

Alternative Splicing:

not all exons are always included in final RNA allows for various combinations of exons

What would be the most likely affect of a nucleotide change in the region of the gene that will be an intron in the mRNA?

nothing

Frameshift causing extensive missense

nucleotide deletion causing the reading frame to be off and for the wrong amino acid sequence to be translated

Nonsense

nucleotide substitution that reads as a stop codon and results in the wrong protein being synthesized

Missense

nucleotide substitution with change in amino acid chain

Polyadenylation mechanism:

occurs at 3' end 1. Two Groups of proteins which recognize the 2 poly (A) signals on pre-mRNA 2. two groups interact to bend the mRNA 3. Recruitment of poly-A polymerase (PAP) 4. Cleavage in poly (A) site allowing tail to fall off 5. PAP begins to synthesize poly (A) tail slowly 6. Helper protein comes in to speed up the process

Tetracycline antibiotics bind to the A site on the bacterial ribosome and prevent further translation. Where would you expect polypeptides to accumulate on tetracycline- inhibited ribosomes?

on a tRNA attached to the P site

During transcription of a particular gene, the RNA polymerase will transcribe:

only one of the DNA strands, moving in a 3' to 5' direction along the template.

Alternative 5' or 3' splice sites

part of exon cut off (not whole included or deleted)

Anticodon

part of tRNA that pairs with mRNA and decides what amino acid goes there 3'-->5'

coupled transcription and translation occurs in what organisms?

prokaryotes

Exon Splicing Enhancer

region with in exon where additional factors can bind to effect splicing

Eukaryotes have mostly transposons or retrotransposons?

retrotransposons

During the processing of introns, a single snRNP complex catalyzes both the cleavage of the RNA and the joining of the cut ends. What would be the consequence if these two processes were catalyzed by separate enzymes not associated in a single complex?

the exons might not be joined in the correct sequence.

in order to be considered a mature RNA in a Eukaryotic system...

the poly(A) tail and 5' cap must be added and the introns must be spliced out

Nuclear Pore Complex

tightly regulates what gets in and out of nucleus

RNA polymerase II:

transcribes: mRNA, snRNA, siRNA, miRNA Function: encodes protein, RNA splicing, chromatin-mediated repression, translation control

RNA polymerase I:

transcribes: pre-rRNA Function: Ribosome components, protein synthesis

RNA polymerase III:

transcribes: tRNAs, rRNA, snRNA, RNA Function: protein synthesis, ribosome component, RNA splicing, signal recognition particle for insertion of polypeptides into the ER, etc.

Are transposons or retrotransposons more common in eukaryotes than prokaryotes?

transposons

Prokaryotes have mostly (DNA transposons OR retrotransposons)?

transposons

Retrotransposon mechanism:

uses "copy and paste" mechanism 1. RNA polymerase II makes an RNA copy from retrotransposon 2. Reverse transcriptase converts RNA intermediate back into DNA 3. Retrotransposon DNA copy is inserted into target DNA

DNA Transposon mechanism:

uses "cut and paste" mechanism. 1. DNA transposon is extracted from donor DNA (Transposase makes blunt-ended cuts in donor DNA and staggered cuts (sticky ended overhangs which match sequence) in target DNA 2. DNA transposon then inserted into target DNA (transposase ligates transposon to 5' single-stranded ends of target DNA) 3. cellular DNA polymerase extends 3' cut ends and ligase joins extended 3' ends to transposon 5' ends

Alternative promoters

where RNA polymerase binds to DNA sequence can vary

What would happen if an intron is inserted into a retrotransposon?

The intron would be lost during retrostransposition

In an experimental situation, a student researcher inserts an mRNA molecule into a eukaryotic cell after she has removed its 5′ cap and poly-A tail. Which of the following processes would you expect her to find to have occurred?

The molecule is digested by enzymes because it is not protected at the 5′ end.

Given what you know about the differences between prokaryotes and eukaryotes, how are the processes of transcription and translation different? Think about the components, localization, and timing.

Euk transcription has a bunch of transcription factors while Pro generally just has a sigma factor helping RNA pol. There is no nucleus in Pro, like in Euk, meaning that transcription and translation can be coupled (happening simultaneously) making it much faster. There also doesn't need to be nuclear export in pro. Pro don't do mRNA processing, including splicing, which also makes the process faster than in euk.

With respect to gene transcription, what does relative expression mean?

It can mean the amount of expression from one gene in comparison to another gene (perhaps expressed as a ratio).

Transposable elements can cause corn kernels to be spotted if they jump in or out of the color genes during the development of the kernel. In both kernels shown below a transposon is disrupting the same gene controlling color. Why are the sizes of the spots so different between the two?

Kernel development consists of many rounds of mitosis. When a transposon jumps out of a disrupted "color gene" in a cell, that cell, and all cells descending from it, will contain a functional copy of the "color gene," creating a sector of purple cells surrounded by yellow cells. The earlier the transposition event occurs in development, the more descendants will arise. Thus, a bigger purple spot will be observed.

Exon Skipping using Tra2 protein

Males: Tra2 bound to ESE--> exon skipped Females: Tra2 bound to ESE--> recruits splicing factors--> exon included