Genome Analysis & Gene Prediction

Overview about GenesGene : whole nucleic acid sequence necessary for the synthesis of a

functional protein (or functional RNA)A human cell contains approximately 23,000 genes. Some of these are expressed in all cells all the time. These so-

called housekeeping genes are responsible for the routine metabolic functions (e.g. respiration) common to all cells.

Some are expressed as a cell enters a particular pathway of differentiation.

Some are expressed all the time in only those cells that have differentiated in a particular way. For example, a liver cell expresses continuously the genes for the metabolizing enzymes.

Some are expressed only as conditions around and in the cell change. For example, the arrival of a hormone (due to environmental factors or others) may turn on (or off) certain genes in that cell.

How Gene Expression is Regulated?

To Know about gene expression, first we look for the basic structure of a gene.

Genomic DNAUpstream Primary

TranscriptDownstreamGenomic

DNA5’…. …3’

About Upstream region of a Gene

Upstream Primary Transcript

Downstream

Upstream promoter/Regulatory region

Promoter

Genomic DNA

Upstream

5’…. …3’

Regulatory Locus

Distal Central Proximal

Distal (GC box) Central (CAAT box)

Core/basal Promoter (TATA Box)

About Core Promoter

basal or core promoter located within about 40 base pairs (bp) of the transcription start site (TSS)

It is found in all protein-coding genes. This is in sharp contrast to the upstream promoter whose structure and associated binding factors differ from gene to gene.

It contains a sequence of TATA box (either canonical TATA box or TATA variant). It is bound by a large complex of some 50 different proteins, including- Transcription Factor IID (TFIID) which is a complex of TATA-binding protein (TBP), which recognizes and binds to the TATA

box 14 other protein factors which bind to TBP — and each other — but not to

the DNA.- Transcription Factor IIB (TFIIB) which binds both the DNA and pol II.

About Upstream Promoter/Regulatory Regions

an "upstream" promoter, which may extend over as many as 200 bp farther upstream

It has three regions- Proximal region: insulators are possibly present in this region. Insulators are stretches of DNA (as few as 42 base pairs) and located between the

enhancer(s) and promoter or silencer(s) and promoter

of adjacent genes or clusters of adjacent genes. Their function is to prevent a gene from being influenced by the enhancer (or silencer) of its neighbors.

- Central Region: Silencers are possibly present in this region. Silencers control regions of DNA that may be located thousands of base pairs away from the gene they control. However, when transcription factors (Silencers) bind to them, expression of the gene they control is repressed.- Distal Region: Enhancers may be present in this region. Enhancer bind to regions of DNA that are thousands of base pairs away from the gene they control. Binding increases the rate of transcription of the gene. Enhancers can be located upstream, downstream, or even within the gene they control.

About Upstream Promoter/Regulatory Regions

About Primary TranscriptUpstream Primary

TranscriptDownstreamGenomic

DNA5’…. …3’

ATG…. GT……..AG

………...

GT…..AG

…......TGA

Exon

Start codon

Exon

Acceptor site

Donor site

Exon

Intron

mRNA

Stop codon

Intron

ATG…………………………………………TGA

TSS

Primary transcript consists of Cap region: 5' cap is a specially altered nucleotide on the 5'

end of precursor messenger RNA. 5’-UTR: Regions of the gene outside of the CDS are called UTR’s

(untranslated regions), and are mostly ignored by gene finders, though they are important for regulatory functions.

Coding sequence (CDS): CDS of a gene is delimited by four types of signals: start codons (ATG in eukaryotes), stop codons (usually TAG, TGA, or TAA), donor sites (usually GT), and acceptor sites (AG).

3’-UTR: three prime untranslated region (3' UTR) is a particular section of messenger RNA (mRNA).

Poly-A tail: Polyadenylation is the addition of a poly(A) tail to an RNA molecule. The poly(A) tail consists of multiple adenosine monophosphates.

About Primary Transcript

About Intron and Exon  Intron: It is derived from the term intragenic

region, i.e. a region inside a gene. these are sometimes called intervening sequences which refer to any of several families of internal nucleic acid sequences that are not present in the final gene product

Exon: these sequences are present in the mature form of an RNA molecule after removing of introns. The mature RNA molecule can be a messenger RNA or a functional form of a non-coding RNA such as rRNA or tRNA.

More about Exon Three types of exons are defined:

initial exons extend from a start codon to the first donor site; internal exons extend from one acceptor site to the next donor site; final exons extend from the last acceptor site to the stop codon;

single exons (which occur only in intronless genes) extend from the start codon to the stop codon.

Structure of a Gene

An Hypothetical Example Gene Parse Tree

Gene Prediction

Analysis by sequence similarity can only reliably identify about 30% of the protein coding genes in a genome

50-80% of new genes that are identified, have a partial, marginal, or unidentified homolog

Frequently expressed genes tend to be more easily identifiable by homology than rarely expressed genes

Gene finding is species-specific

Codon usage patterns vary by species Functional regions (promoters, translation

initiation sites, termination signals) vary by species

Common repeat sequences are species-specific

Gene finding programs rely on this information to identify coding regions

Protein Coding Gene ab initio using computational

methods is the most suited to protein-coding genes

Protein-coding genes have recognizable features• open reading frames (ORFs)• codon bias• known transcription and translational start

and stop motifs (promoters, 3’ poly-A sites)

• splice consensus sequences at intron-exon boundaries

ab initio gene discovery• Protein-coding genes have recognizable

features• We can design software to scan the genome

and identify these features• Some of these programs work quite well,

especially in bacteria and simpler eukaryotes with smaller and more compact genomes

• It’s a lot harder for the higher eukaryotes where there are a lot of long introns, genes can be found within introns of other genes, etc.

ab initio gene discovery—Validating predictions and refining gene models

Standard types of evidence for validation of predictions include:• match to previously annotated cDNA• match to EST from same organism• similarity of nucleotide or conceptually translated

protein sequence to sequences in GenBank• protein structure prediction match to a PFAM

domain• associated with recognized promoter sequences, ie

TATA box, CpG island• known phenotype from mutation of the locus

Finding Non–protein Coding Genes

• Non-protein coding genes (tRNA, rRNA, snoRNA, siRNA, miRNA, various other ncRNAs) are harder to find than protein-coding genes. Because• often not poly-A tailed—don’t end up in

cDNA libraries• no ORF• constraint on sequence divergence at

nucleotide not protein level, so homology is harder to detect

To find out, Non-protein coding genes, we have identify…..

• secondary structure• homology, especially alignment of related

species• experimentally

• isolation through non-polyA dependent cloning methods

• microarrays

Finding Non–protein Coding Genes

Most gene-discovery programs makes use of some form of machine learning algorithm. A machine learning algorithm requires a training set of input data that the computer uses to “learn” how to find a pattern.

Two common machine learning approaches used in gene discovery (and many other bioinformatics applications) are Dynamic programming model Artificial neural networks (ANNs) and Hidden Markov models (HMMs)

ab initio gene discovery—approaches

Control of Gene Expression—Transcription Factors

Transcription factors (TFs) are proteins that bind to the DNA and help to control gene expression. The sequences to which they bind are transcription factor binding sites (TFBSs), which are a type of cis-regulatory sequence

Most transcription factors can bind to a range of similar sequences. These can be found in either of two ways, as a consensus sequence, or as a position weight matrix (PWM).

Once we know the binding site, we can search the genome to find all of the (predicted) binding sites

Evidence based Approaches Comparative or similarity based

gene prediction Combine gene models with

alignment to known ESTs & protein sequences

Gene Prediction Tools SNAP TwinScan Gnomon (NCBI) GeneWise Jigsaw GLEAN Grail

BLAST FASTAX BLAT WABA MZEF, MZEF-SPC FGENESH

Genome Annotation-Much work remains

Despite good progress in identifying both protein coding and non-protein coding genes, much work remains to be done before even the best-studied genomes are fully annotated.

For the higher eukaryotes, only a tiny percentage of features such as TFBSs and other non-gene features have so far been indentified.

References http://users.rcn.com/jkimball.ma.ultra

net/BiologyPages/P/Promoter.html