Unit 2

gene expression

term for the processes by which cells produce functional products

gene regulation

the term for the processes that control gene expression

epigenetics

term for stable changes to DNA not explained by changes in the sequence

DNA methylation

one of the 2 ways epigenetic changes can happen; the addition of methyl groups to cytosines

histone modification

of the 2 ways epigenetic changes can happen; post-translational modification of histone proteins that controls DNA-histone interactions to modulate gene expression

DNA methyltransferases

what are DNMTs?

DNMT1

type of DNMT that maintains methylation patterns already present

DNMT3

type of DNMT that makes new methylation marks

CpG islands

where is methylation enriched?

CpG islands

sequences with many CG dinucleotides, most commonly found in promoters. Methylation is enriched here

promoters

Where are CpG islands most commonly found?

TET enzymes

what enzymes perform demethylation?

promoter

what region of the DNA strand does methylation usually result in a decrease of transcription?

coding DNA sequence

what region of the DNA strand does methylation usually cause an increase in transcription?

transposons

____ are methylated to decrease their activity

bisulfite conversion

method to study DNA methylation by treating a DNA sample with sodium bisulfite

uracil

in a bisulfite conversion, unmethylated cytosines become what?

differentially methylated regions

DMRs (identifying these is the main goal of bisulfite conversion)

nucleosome

made up of DNA and histones

post translational modification

addition or removal of chemical groups on proteins to modify their structure and/or function

8

how many histone proteins form a nucleosome?

H2a, H2b, H3, H4

list the 4 different pairs of histone proteins that bind DNA nonspecifically

H1

histone protein that anchors or holds everything together

lysine, argenine

what 2 positively charged amino acids mostly make up histones?

acetylation

addition of an acetyl group

histone acetyltransferase

enzyme that performs acetylation (HAT)

increase

does acetylation increase or decrease gene expression?

histone deacetylase

enzyme that removes acetyl groups from histones (HDAC)

methylation

addition of a methyl group

histone methyltransferase

adds a methyl group (HMT)

histone demethylase

removes a methyl group (HDM)

3

how many methyl groups can be added at a time?

phosphorylation

addition of phosphates

histone kinase

enzyme that adds a phosphate

histone phosphatase

enzyme that removes a phosphate

ubiquitination

addition of ubiquitin

SUMOylation

addition of small ubiquitin like modifier proteins

small ubiquitin like modifier proteins

SUMO

citrullination

when argenine is irreversibly changed to citrulline

chromatin immunoprecipitation sequencing

what is ChIP-seq?

transcription

synthesis of RNA from a DNA template; produces RNA complementary to one of the DNA strands

RNA polymerase

what performs transcription?

ribosomal RNA

what RNA does DNA pol 1 transcribe?

messenger RNA, micro RNA

which 2 RNA does RNA pol 2 transcribe?

transfer RNA

which RNA does RNA pol 3 transcribe along with other small RNAs?

transcriptome

the collection of RNA in a sample that causes changes in gene expression

polymerases

____ bind the template strand and assemble transcripts “identical”* to the coding strand (T-U)*

initiation, elongation, termination

list the three steps of transcription in order

transcriptional initiation

step where RNA polymerase and general transcription factors bind promoter

general transcription factors

a transcription factor involved in transcribing most or all of the genes transcribed by a specific polymerase

TATA box

name for the actual nucleic acid sequence at the promoter

TF2D

general transcription factor that binds first; other TFs bind to for the pre-initiation complex

pre initiation complex

group of TFs that bind at the promoter

transcriptional elongation

when RNA polymerase builds the RNA transcript

transcriptional termination

when RNA polymerase stops at the polyadenylation sequence (PAS)

polyadenylation sequence

another term for the 3’ poly A tail (PAS)

cleavage and polyadenylation specific factor

CPSF, enzyme that cleaves the RNA after the PAS

mRNA

_____ must be processed after transcription

primary transcript

name for the product of transcription AKA pre-mRNA

introns

what gets spliced out of mRNA?

7-methylguanosine

modified nucleotide required for stable mRNA durning translation, provides resistance to exonucleases. this is the 5’ cap

exonucleases

enzymes that break down nucleic acids

first

during the ____ step of the 5’ mRNA capping process, a 5’ phosphate is removed by RNA triphosphatase

second

during the ____ step of the 5’ mRNA capping process, GTP is added to the 5’ end by mRNA guanylyltransferase forming a 5’-5’ linkage

third

during the ____ step of the 5’ mRNA capping process, the guanine is methylated to form 7-methylguanosine by the enzyme mRNA cap guanine-N7 methyltransferase or RNMT

yes

can there be a fourth step of the 5’ mRNA capping process?

RNA triphosphatase

what enzyme removes a 5’ phosphate during the first stage of the 5’ mRNA capping process

mRNA guanylyltransferase

what enzyme adds a GTP to the 5’ end of mRNA during the second stage of the 5’ mRNA capping process?

mRNA cap guanine-N7 methyltransferase

what enzyme methylates guanine to for 7-methylguanosine? aka RNMT

spliceosome

splicing is performed by the ____; exons are expressed and introns are removed

small nuclear ribonucleoproteins

made up of proteins and small nuclear RNAs (snRNAs) aka snRNPs. Recognize splice sites by base pair

GT, AG

introns almost always start with “____“ (donor site) and end with “____“ (acceptor site)

snRNPs

what is the spliceosome is made up of? abv

U1, U2, U6

what are the 3 snRNPs we talked about in class?

U1

of the 3 snRNPs that we talked about in class, this one recognizes the donor site

U2

of the 3 snRNPs that we talked about in class, this one recognizes the acceptor site

U6

of the 3 snRNPs that we talked about in class, this one double checks U1s binding, then works with U2 to cut out introns using ATP hydrolysis

alternative splicing

term for the concept that one transcript can have multiple splicing patterns.

isoforms

multiple protein or transcript forms a gene can have

polyadenylation

the addition of a 3’-poly (A) tail to a mRNA transcript. Begins during transcriptional termination when CPSF binds the ____ site, recruits CstF for RNA cleavage and poly (A) addition

AAUAAA

what is the sequence of the polyadenylation site?

polyadenylate polymerase

enzyme that performs poly (A) addition aka PAP

alternate polyadenylation

term for the concept that poly (A) tails can have multiple lengths

poly (A) binding protein

binds poly (A) tail; promotes nuclear export and translational initiation aka PABP

adenosine deaminase

enzyme that changes adenosine to inosine aka ADAR

environmental, cell signaling, cell type, developmental period

list the 4 factors that effect gene expression mentioned in class

serial analysis of gene expression

“old school” transcriptomic method that uses expressed sequence tags (RSTs, coding sequence fragments) then sanger sequencing. aka SAGE

DNA microarrays

“old school” transcriptomic method that uses “chips“ with DNA fragment. The experimental DNA is added and will base pair with the chip DNA, the amount of binding is measured by fluorescence, and requires sequence of interest to be on the chip

expressed sequence tags

coding sequence fragments used in serial analysis of gene expression (SAGE) aka ESTs

RNA-seq

____ is the most common transcriptomic method currently used

obtain RNA, break up RNA, make cDNA, add adapters to cDNA, sequence cDNA, mapping to reference

list the 6 steps of RNA-seq in order

complementary DNA

cDNA

mRNA-seq

term for only mRNA sequencing

totalRNA-seq

term for sequencing all RNA (except ribosomal usually)

spacial transcroptomics

sequencing a single cell or single tissue/ cell type RNA

quality control, pre-processing, alignment, assembly, quantification, differential analysis, annotation

list the 7 steps of the RNA-seq workflow in order

FASTQ

In the first step of the RNA-seq workflow (after sequencing reactions) what tool did we use in lab for quality control and to output Phred scores?

pre-processing

what is the second step of the RNA-seq workflow (after sequencing reactions) where you trim and filter reads, remove adapters and low quality sequences

featureCounts

in the fifth or the quantification step of the RNA-seq workflow (after sequencing reactions), what tool did we use to measure up regulation and down regulation

differential analysis

the sixth step of the RNA-seq workflow (after sequencing reactions) where we look for what is significantly up/down